Genomic Signal Analysis of Avian Influenza Virus Variability

Transcription

1 Genomic Signal Analysis of Avian Influenza Virus Variability Paul Dan Cristea, Senior Member, IEEE, Rodica Tuduce, Member, IEEE Abstract The paper gives a brief presentation of the avian influenza, of the previous worldwide outbursts caused by type A viruses, and presents aspects of Orthomyxoviruses structure that determines their pathological features and the current epidemiologic threats. Nucleotide sequences of the genes that compose the genome of the H5N1 avian influenza subtype, downloaded in symbolic form from the GenBank of NIH [1], have been converted into complex digital genomic signals. The variability of the viruses is studied by analyzing the changes of the cumulated phase of the genomic signals for the eight segments of the viral genome. A I. INTRODUCTION LL avian influenza viruses are type A influenza viruses in the family of Orthomyxoviridae [2]. All known strains of the influenza A virus are carried by and can infect birds [3]. Orthomyxoviruses are the agents of several other similar conditions named "flu" or "grippe", found both in humans and animals [4]. In humans, these are viral infections that attack mainly the respiratory tract, are transmitted from person to person by saliva droplets expelled by coughing and cause symptoms that include fever, headache, fatigue, sore joints, dry cough, sore throat, nasal congestion, sneezing, irritated eyes, and severe muscle aches. In the elderly, young child and infirm, influenza is a major cause of disability and death, often combined with a secondary infection of the lungs by bacteria. This association favored the confusion of influenza with other viral or bacterial infections that cause fever and sniffles and keep the patient in bed for one or several days, or even with some gastrointestinal upsets, the so called "stomach flues". The early origin of the term influenza was in the belief that the disease was caused by unfavorable astrological "influences". In the 18th century, the name was linked to the belief that the disease was caused mainly by the "influence of the cold". Before viruses have been discovered, the Haemophilus influenzae bacterium, frequently found in the lungs of affected people, has been considered the main pathogen and given the name of the disease. As mentioned, this and other bacteria such as Staphylococcus aureus or Streptococcus pneuminiae cause opportunistic secondary infections of the lung. Only in 1930 in pigs and 1933 in humans it was established that influenza is caused by a virus. Influenza was responsible for what is now known as the most devastating plague in human history the "Spanish Manuscript received December 28, The work has been partially supported by grants from the Ministry of Education and Research of Romania in the framework of the RELANSIN Programme 2170/ , the CNCSIS Programme project / , CEEX Programme project 50/2005. P. D. Cristea and R. Tuduce are with the Biomedical Engineering Center of the University "Politehnica" of Bucharest, Spl. Independentei 313, , Bucharest, Romania (phone: ; fax: ; pcristea@dsp.pub.ro). flu" pandemic of , which infected about 500 million and killed between 20 and 50 million people worldwide [5, 6]. There has never been as high mortality in a small period of time in world's history, including both World Wars. More people died of influenza in a single year than in four-years ( ) of Black Death Bubonic Plague. It has not been exactly established where the 1918 flu pandemic started from, probably in China as the result of a rare genetic shift of the influenza virus, but it certainly was not in Spain. In pockets across the globe, a new type of flu erupted, initially seemingly as benign as the common cold. However, the influenza of that season was the first large scale attack on humans of the influenza type A virus, which till then stayed mainly in birds, its normal hosts. Preceding human influenza cases where caused mainly by B and C type viruses, normally found only in humans. Although influenza type B viruses can cause human epidemics, they have never caused a pandemic, whereas influenza type C viruses cause only mild illness and no epidemics or pandemics. After a genetic shift, avian type A virus became able to pass to humans, who had no previous immunity for this type of virus [4, 7]. In the two years of the Spanish flu disaster, a fifth of the world population was infected. Astonishedly, the flu was most deadly for people of ages 20 to 40, a completely unusual pattern of morbidity for influenza. Half of the soldiers who died in Europe during the First World War fell to the influenza virus and not to the enemy. The effect of the influenza pandemic was so severe that in most countries, including US, the average life span was depressed by 10 years. The influenza virus of 1918 had a very high virulence, with a mortality rate of 2.5% 5%, more than twenty times higher than in previous influenza epidemics. Influenza A is responsible for all large flu outbreaks, including the three pandemics/epidemics in (Spanish Flu), (Asian Flu, million dead), and (Hong Kong Flu, 1 million dead). Wild birds, primarily aquatic birds, are the natural asymptomatic hosts of the avian flu virus [3, 8, 9]. Birds are chronically infected with many flu viruses simultaneously and carry the viruses in their intestines, but typically do not get sick from it. However, bird flu is very contagious among birds and can make some domesticated birds very sick and even kill them. The known transmission paths of type A viruses include, apart of the contagion among wild and domestic birds, the transmission from wild birds to humans [10] and other mammals, including pigs, horses, seals, whales, in special circumstances. Transmission has also been proven between poultry and humans, or between pigs and humans, but other paths, such as poultry to horse, seem impossible. The transmission of avian flu virus has also been shown to happen from chicken to tigers, leopards, domestic cats and mice who were fed uncooked poultry [11].

2 It is currently believed that an influenza pandemic could be triggered by a double infection, when an avian-adapted virus and a human-adapted virus infect simultaneously the same host, human or porcine. Viruses may then recombine within the host, in a process called reassortment, to form a genetically new virus able to infect humans and be transmitted efficiently from person to person [10]. II. STRUCTURE AND PATHOGENICITY OF TYPE A FLU VIRUSES The structure of a type A influenza virus is schematically shown in Fig. 1. The shape is roughly spherical and about 200 nm in diameter. The virion has an outer protective capside containing about 3000 molecules of matrix proteins, enveloped by a lipid bilayer derived from the cytoplasmic membrane of the host cell. To be complete, thus infectious, the virion particle needs the lipid envelope, but this makes it quite vulnerable to heat, acidity and lipide solvents. The envelope embeds two specific antigenic glycoproteins anchored in the matrix protein and projecting out of the virion surface, the Hemagglutinin (HA) and the Neuraminidase (NA). Each virion has about 500 HA and about 100 NA molecules on its surface. The glicoproteins determine the serologic specificity of the different subtypes of influenza type A virus and are used for their classification. At least sixteen different types of HA proteins [1, 12] and nine different types of NA proteins are currently known [1]. Many different combinations of HA and NA proteins are possible, but only the H1N1 (Spanish endemic), H1N2 (Asian epidemic), and H3N2 (Hong Kong epidemic) have largely circulated among humans [3,4,7-9]. The H2N2 virus has circulated in humans at the beginning of Asian epidemic, and has caused annual epidemics until 1968, when it vanished after the emergence of H3N2 viruses. Influenza in humans caused by other subtypes have also been reported: H5N1 (122 cases, 62 deaths, outside China by November 2005), H7N7 (Netherlands, 2003, 89 cases, 1 death), H9N2 (China and Hong Kong, 1999, 2003, 3 cases, fully recovered), H7N2 (US, , 2 cases, fully recovered), H7N3 (Canada, 2003, 2 cases, fully recovered). Other subtypes are found in various animal species, mostly in birds, but also in swine, horses (H7N7 and H3N8) and many other species. The current flu threat is in the highly pathogenic H5N1 subtype [10,13-16], which has been detected in wild migratory waterbirds and has spread among domestic poultry. The virus remains still avian-adapted, but it has been found that single amino acid changes in this avian virus strain type H5 hemagglutinin make it able to infect humans. The current H5N1 virus does not propagate from person to person. But, if this virus becomes human-adapted by an antigenic shift, a pandemic may be unavoidable since there will be very little immunity to this genetically new virus. The hemagglutinin of the H5N1 virus has been associated with the high pathogenicity of this flu virus strain. Hemagglutinin recognizes the cells that the virus can invade [17,18]. HA selectively binds to the sialic acid of the Fig. 1. Schematic representation of a virus from the orthomyxoviridae family. The genome is divided into eight distinct RNA single strands associated with nucleoproteins. The protective envelope embeds two specific antigenic glycoproteins, hemagglutinin and neuraminidase. host cell surface receptors. The HA molecule is composed of 550 amino acids and is 135 Angstroms long. The name hemagglutinin comes from HA ability to cause the agglutination of erythrocytes [Nelson 2005]. There are five known epitopes in the HA1 region of HA, where antibodies bind during the immune response, triggering the antigenic drift variability. Neuraminidase enables the release of new viruses from the infected cells, helps the virus to pass through mucous between cells in the entire respiratory tract, and cleaves sialic acid residues from viral proteins, preventing the aggregation of viruses [18]. NA consists of a single polypeptide chain that is oriented in the opposite direction with respect to HA and projects as a mushroom-shape spike on the virus surface. Administration of chemical inhibitors of neuraminidase, such as zanamivir and oseltamivir, can prevent progeny viruses from escaping infected cells, therefore limiting the severity and spread of viral infections [19]. A typical feature of influenza A viruses is that its genome is divided in eight distinct linear segments of negative-sense single stranded RNA, schematically shown in Fig.1. The segmented nature of the genome increases the probability of an antigenic shift, by favoring the abrupt variability by exchange of entire genes between different viral strains cohabitating the same cell. Each segment contains a single gene, but some of them generate two distinct proteins by using different reading frames. The genome comprises: (1) HA gene, (2) NA gene, (3) NP gene, (4) M gene, (5) NS gene, (6) PA gene, (7) PB1 gene, and (8) PB2 gene. The NP gene encodes the nucleoprotein. Influenza viruses types A, B, and C are distinguished by their nucleoproteins. The M and NS genes encode each two matrix proteins (M1, M2) and two non-structural proteins (NS1, NS2), respectively, by using different reading frames. The PA [20], PB1, and PB2 genes encode the influenza virus RNA polymerase, a heterotrimeric protein, which is a RNA dependent RNA replicase. Each RNA strand is complexed with several copies of NP to form a NP-complexed viral RNA (vrna). RNA polymerase is a heterotrimer composed

3 from the PB1, PB2, and PA subunits. It associates with vrna, and with some "non-structural" (NS1, NS2) protein molecules, to form a virion ribonucleoprotein (vrnp). The negative-sense RNA strands need to be transcribed into positive-sense RNA strands and to get a cellular 5' cap added in the host cell to become active, i.e., to be processed as a mrna by the ribosomes. The positive-sense RNA strands also serve for the synthesis of new negative-sense RNA strands for the replication of virions. Thus, vrnp directs two types of RNA synthesis: mrna synthesis (transcription) and vrna amplification (replication). The negative-sense RNA strategy has the advantage of providing many copies of mrna, but requires the virion to have its own RNA replicase that does not exist in the host cell. The cycle of the influenza virus attack passes through the following steps: (1) viral hemagglutinin molecules bind to carbohydrate on the glycoproteins of the epithelial cells of the host, (2) virion is engulfed by receptor mediated endocytosis, (3) structure of the hemagglutinin changes, enabling it to fuse the viral membrane to the vesicle membrane, (4) virus content is put in contact with the cytosol, (5) RNA synthesis and its assembly with NP takes place in the cell nucleus, (6) synthesis of proteins takes place in the cytoplasm, (7) virion core leave the nucleus and migrates towards the cell membrane, together with hemagglutinin, neuraminidase, M1 and M2 proteins, buds through the viral protein patches, and releases new viruses. Amantadine and Rimantadine antiviral drugs, can shorten the duration and moderate the severity of influenza by inhibiting the viral replication, possibly by interfering with the M2 viral protein that is needed for uncoating the virion when it enters an infected cell [21, 22]. Because of the variability of the influenza virus caused by the continuous antigenic drift, between the jumps caused by antigenic shifts, the strains used to prepare vaccines must be adapted every season, to be kept active against newly emerging strains. Till now, antigens from H1N1, H3N2 and from a type B virus have been used for vaccines. The current vaccine is based on A/New Caledonia/99, A/California/2004, and B/Shanghai/2002. Fig. 2. Number of the influenza virus HxNy subtype sequences in the GeneBank [1] database on December 20 th, III. GENOMIC SIGNALS OF AN AVIAN INFLUENZA VIRUS TYPE A, SUBTYPE H5N1 Nucleotide sequences that compose the genome of Influenza virus Type A have been downloaded from NIH Genbank database [1], for all the subtypes specified in Fig.2. The symbolic sequences have been converted into (complex) digital genomic signals using the procedure previously described in [23]. Phase analysis has been performed on the resulting genomic signals. The cumulated phase measures the unbalance in the distribution of nucleotides along a nucleic acid strand (statistics of first order): 3(n G n C ) + (n A - n T ), where n G, n C, n A, and n T give the number of occurrences of guanine, cytosine, adenine, and tymine, respectively. The unwrapped phase measures the unbalance between the number of direct and inverse nucleotide transitions (n + - n - ), describing a second order statistical feature of the sequence. In this paper, results referring to the cumulated phase of the genomic signals for the eight nucleotide sequences that compose the genome of subtype H5N1 viruses isolated in HongKong in several distinct instances, are presented. The samples include genomes of viruses isolated from humans and chickens in a period from 1997 to Fig. 3. presents the cumulated phase of twelve genomic signals of the hemagglutinin (HA) gene of the Influenza virus type A, subtype H5N1, the strains with accessions AB212054, AF046080, 88, 96, 97, 99, AF082034, 35, 37, AF102680, 81, 82 [1, 14, 24-26]. The effect of the continuous pressure of the genetic drift is reflected in the high variability of the HA gene. Despite the clear similarities in large segments of the signals, the 12 samples are structured in three quite distinct clusters, that correspond to the different sources and dates of the material. The cumulated phase diagrams for the three genomic signals of the neuraminidase (NA) gene, with accessions AB212056, AF046081, 9 [1, 14, 24], shown in Fig. 4, are grouped in two clusters that correspond to the two available sources of data. As in the case of other genomic signals, the variations of the sequence, thus of the associated signals, take place mainly in certain specific sites along the gene strand and the variations tend to compensate [27, 28]. Significant variations are evident for the nucleoprotein (NP) gene in Fig. 5, the matrix protein (M1) gene in Fig. 6, and the nonstructural protein NS1 gene in Fig. 7. From the three parts of the polymerase gene, the highest variation occurs in the acidic component PA in Fig. 8, while the two basic components PB1 and PB2, in Figs. 9 and 10, respectively, vary quite little, for the same virions. Similar to the case of other viruses, the unwrapped phase (not shown) has larger variations than the cumulated phase [24, 25]. This is caused by the fact that drift mutations, occurring continuously, are of the single nucleotide polymorphism (SNP) type, while the crossover mutations take place only as effect of much less frequent and more dangerous genetic shift events, as reassortments. It has been shown that unwrapped phase is sensitive to SNPs, but insensitive to crossover [26].

4 Fig. 3. Hemagglutinin (HA) gene (accessions AB212054, AF046080, 88, 96, 97, 99, AF082034, 35, 37, AF102680, 81, 82 [1, 14, 27-29]). Fig. 7. Nonstructural protein (NS1) gene (AB212058, 59, AF046083, 91 [1]). Fig. 4. Neuraminidase (NA) gene (AB212056, AF046081, AF [1]). Fig. 8. RNA polymerase PA gene (AB212053, AF046087, AF [1]). Fig. 5. Nucleoprotein (NP) gene (AB212055, AF046084, AF [1]). Fig. 9. RNA Polymerase PB1 gene (AB212052, AF046085, AF [1]). Fig. 6. Matrix protein (M) gene (AB212057, AF046082, AF [1]). Fig. 10. RNA polymerase PB2 gene (AB212050, 51, AF046086, 93 [1]).

5 IV. CONCLUSIONS The conversion of nucleotide sequences into digital signals offers the possibility to apply signal processing methods for the analysis of genomic data. A detailed description of the complex genomic signal representation used in our work can be found in [23]. This method has already proven its potential both in revealing large scale features of DNA sequences, including both coding and noncoding regions, at the scale of whole genomes or chromosomes [30, 31], but also in studying the variability of pathogens, e.g., the Human Immunodeficiency Virus, type 1 (HIV-1) [24, 25]. In this paper, we apply the genomic signal approach for studying the variability of the H5N1 avian influenza virus. We report first results in this domain, using only the cumulated phase of the complex genomic signals for the eight nucleotide segments of the viral genome. Further work will be focused on the dynamics of all Influenza Type A viruses that have crossed till now the species barrier from birds to humans, and which hold the potential to become highly contagious and highly lethal in humans, including the H5N1 subtype. Phylogenetic neighbor-joining trees of the viral genes will also be carried out, especially for the highly variable genes. Variability signals with respect to average, median and maximum flat references, and the digital derivatives of genomic signals will also be applied as described in [25]. REFERENCES [1] National Center for Biotechnology Information, National Institute of Health, National Library of Medicine, National Center for Biotechnology Information, GenBank, [2] E. Fodor, G.G. Brownlee, in Influenza, ed. C.W. Potter, pp.1-29, Elsevier, Amsterdam, [3] World Health Organization (WHO), [4] N.J. Cox, K.Subbarao, Global epidemiology of influenza: past and present, Annu. Rev. Med., vol. 51, pp , [5] A.H. Reid, T.G. Fanning, T.A. Janczewski, R. Lourens, J. K. Taubenberger, Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment, J. Virol., 78, pp , [6] K. Jeffery et.al., Characterization of the 1918 influenza virus polymerase genes, Nature, vol 437, pp , October 2005, (doi: /nature04230). [7] E. Ghedin, N. Sengamalay, M. Shumway et. al., Large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution, Nature, vol. 437, 20 Oct 2005, pp (doi: /nature04239). [8] The World Health Organization Global Influenza Program Surveillance Network, Evolution of H5N1 avian influenza viruses in Asia, in Emerging Infectious Diseases (2005). [9] [10] The Writing Committee of the World Health Organization (WHO), "Avian Influenza A (H5N1) Infection in Humans," New England Journal of Medicine, vol. 353, pp , 29 September [11] T. Kuiken et al, Avian H5N1 Influenza in Cats, Science, vol. 306, pp. 241, (doi: /science ). [12] R.A. M. Fouchier et.al., Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls, J. Virol, vol 79 (5), pp , [13] M. Hatta et.al, Molecular Basis for High Virulence of Hong Kong H5N1 Influenza A Viruses, in Science, vol. 293, pp , [14] D.L. Suarez,.et.al., Comparisons of highly virulent H5N1 influenza A viruses isolated from humans and chickens from Hong Kong, J. Virol., vol. 72 (8), pp , (AF046084) (AF046092) [15] M. Enserink, Avian Influenza: 'Pandemic Vaccine' Appears to Protect Only at High Doses," Science, vol 309, pp. 996, 12 August 2005 (doi: /science b) [16] D.A. Senne et.al, Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the HA cleavage site as a marker of pathogenicity potential, Avian Disease, vol. 40, pp [17] J.M. White et al, Attachment and entry of influenza virus into host cells. Pivotal roles of hemagglutinin, in Structural Biology of Viruses, W. Chiu, R.M. Burnett, R.L. Garcea, editors. Oxford University Press, NY. pp80-104,1997. [18] Y. Suzuki, Sialobiology of Influenza: Molecular Mechanism of Host Range Variation of Influenza Viruses, Biological and Pharmaceutical Bulletin, vol. 28, pp , [19] F.G. Hayden et.al., Use of the selective oral neuraminidase inhibitor oseltamivir to prevent influenza, N Engl J Med, pp.341, 1999, pp , PMID [20] B. Perales, J. Sanz-Ezquerro, P. Gastaminza, The Replication Activity of Influenza Virus Polymerase Is Linked to the Capacity of the PA Subunit To Induce Proteolysis, J. Virol, vol 734 (3), pp , [21] T. Jefferson et.al., Amantadine and rimantadine for preventing and treating influenza A in adult,. Cochrane Database Syst Re, vol.3, 2004, CD001169, PMID [22] R.A. Bright, M.J. Medina, X. Xu et.al., Incidence of adamantane resistance among influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: A cause for concern,, Lancet, vol.366 (9492), pp [23] P. D. Cristea, Representation and Analysis of DNA sequences, in Genomic Signal Processing and Statistics, Editors E.G. Dougherty, I. Shmulevici, Jie Chen, Z. J. Wang, Book Series on Signal Processing and Communications, Hidawi, 2005, pp [24] P. D. Cristea, D. Otelea, Rodica Tuduce, Genomic signal analysis of HIV variability, in Proc. SPIE, BIOS 2005, vol. 5699, paper 52. [25] P. D. Cristea, Genomic Signal Analysis of HIV-1 Clade F Gene Variability, invited paper, EUROCON 2005 Conference, Sava Center, Belgrade, November 21-24, [26] P. D. Cristea, Invariants of DNA Genomic Signals, SPIE - AU 104 International Conference on Biomedical Applications of Micro- and Nano-engineering, University of New South Wales, Sydney, Australia, December 13 15, 2004, vol. 5651, published in Progress in Biomedical Optics and Imaging, Biomedical Applications of Microand Nano-engineering II, Editor/Chair D. V. Nicolau, Vol. 5 (34), ISSN , 2005, pp [27] K. Shinya, Characterization of a Human H5N1 Influenza A Virus Isolated in 2003, J. Virol., vol. 79 (15), pp , (AB212055). [28] M. Matrosovich et.al., The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties, J. Virol. 73 (2), pp , (AF082034). [29] C. Bender, Characterization of the surface proteins of influenza A (H5N1) viruses isolated from humans in , Virology, vol.1 (254), pp , (AF102680) [30] P. D. Cristea, Large Scale Features in DNA Genomic Signals, ELSEVIER, Signal Processing, Special Issue on Genomic Signal Processing, 83, pp , [31] P. D. Cristea, Genomic Signals of Re-Oriented ORFs, EURASIP Journal on Applied Signal Processing, Special Issue on Genomic Signal Processing, vol. 2004, no.1, pp , January 1, 2004.