Avian Influenza Jen-Ren Wang, Ph. D. (王貞仁) Dept. of Medical Laboratory Science and Biotechnology National Cheng Kung University

Influenza viruses Orthomyxoviridae Influenzavirus A, B, C Genome consists of 8 single-stranded RNA segments Enveloped virus with hemagglutinin and neuraminidase spikes (Linda Stannard, University of Cape Town, S.A.)

Prevalence of human influenza viruses B H3N8 H3N2 H2N2 H2N? H1N1 H1N1 1880 1890 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 Year Hampson, et al. MJA 2006

Pandemics in this century Year Virus subtype Deaths 1918-1919 Spanish " flu" (H1N1) 20-40 million 1957-1958 Asian " flu " (H2N2) 1-2 million 1968-1969 Hong Kong " flu " (H3N2) 700,000 1977-1978 Russian " flu " (H1N1) ? Benign pandemic

Influenza virus A genome Polypeptide Nucleotide Length (Nt) Function PB2 2341 Transcriptase: cap binding PB1 Transcriptase: elongation PA 2233 Transcriptase: protease activity HA 1778 Hemagglutinin NP 1565 Nucleoprotein: RNA binding; part of transcriptase complex; nuclear/ cytoplasmic transport of vRNA NA 1413 Neuraminidase: release of virus M1 1027 Matrix protein: major component of virion M2 Integral membrane protein – ion channel NS1 890 Non-structural: nucleus; effects on cellular RNA transport, splicing, translation. Anti-interferon protein NS2 Non-structural: nucleus+cytoplasm, function unknown Except for proteins in the envelope, there were still several in influenza virus genome. PB1,2,PA protein have the functions of transcriptase. NP was Nucleoprotein. M1 was the major component of virion and NS1 protein has the effects on RNA transport, splicing, and translation. Using these protein, the influenza virus can infect and replicate in cells.

Influenza virus A HA and NA Hemagglutinin(HA) Neuraminidase(NA) Mediating virus binding to cell receptor Promoting release of virus RNP through membrane fusion Posttranslational proteolytic activation of the precursor HA0 into HA1 and HA2 Allows for penetration through RNPs mucous layer to epithelial cells Facilitates release of virions by cleaving sialic acid residues thus preventing aggregation First, let’s summary the function of influenza HA and NA protein. First, HA mediates virus binding to cell receptor, promotes release of virus RNP through membrane fusion, and actives posttranslational proteolysis of the precursor HA0 into HA1 and HA2. Second, Neuraminidase allows for penetration through RNPs mucous layer to epithelial cells and help the release of virions by cleaving sialic acid residues thus preventing aggregation. So, both of these two protein are two most important antigenicity surface glycoproteins in influenza virus.

Molecular determinants of host range restriction Avian Viruses: N-acetylsialic acid α2,3 linked to galactose HA226Gln Human Viruses: N-acetylsialic acid α2,6 linked to galactose HA226Leu By antigenic shift and drift, influenza viruses change their characteristics to infect other species. However, there are still species barriers to stop the spread of viruses. These barriers includes the linkage of N-acetylsialic acid to galactose and hemagglutinin amino acid residues. If the avian influenza virus overcome these barriers, it could infect humans through pigs or directly infect humans. Pigs provide a mixing pot thus allowing the passage of avian virus to humans.

Ecology of influenza A viruses and interspecies transmission Malik Peiris, et al. Clin Micro Rev 20:243, 2007

Avian Influenza Infections in Humans 1997: In Hong Kong, avian influenza A (H5N1) infected both chickens and humans. This was the first time an avian influenza virus had ever been found to transmit directly from birds to humans. 1999: In Hong Kong, cases of avian influenza A H9N2 were confirmed in 2 children. 2003: Two cases of avian influenza A (H5N1) infection occurred among members of a Hong Kong family that had traveled to China. 2003: Avian influenza A (H7N7) infections among poultry workers and their families were confirmed in the Netherlands 2003: H9N2 infection was confirmed in a child in Hong Kong. 2004: H5N1 infections in Asia 2005-present: H5N1 infections in Asia, Europe, others

Avian Influenza (H5N1) Chicken: characterized by sudden onset, severe illness, and rapid death, with a mortality that can approach 100%. Human cases fever sore throat cough fatal cases: severe respiratory distress secondary to viral pneumonia

Avian Influenza (H5N1) Ducks may be infected without illness - 1999-2002, no mortality in ducks - 2002, started causing mortality in ducks Transmitted long distance by migratory birds Pig have been infected (China, Indonesia; asymptomatic) Infect domestic cats, tigers Continue to evolve

Influenza A virus (H9N2) Caused increased morbidity and mortality of chickens and decreased egg production 2 children were infected with respiratory disease Contains 6 genetic segments that are similar to those of H5N1 1999, 2003

Influenza A virus (H7N7) 2003, Netherlands Acute conjunctivitis 430 (380 conjunctivitis, 85 ILI, 56 other) 87 H7 positive conjunctival swab, 10 H7 positive throat swab One fatal case Human-to-human transmission reported

Human disease caused by interspecies transmission of avian influenza viruses without prior reassortment Malik Peiris, et al. Clin Micro Rev 20:243, 2007

Confirmed human cases of H5N1 reported to WHO Country   2003 2004 2005 2006 2007 Total cases deaths Azerbaijan 8 5 Cambodia 4 2 1 7 China 13 3 25 16 Djibouti Egypt 18 10 20 38 15 Indonesia  55 45 31 27 106 85 Iraq Lao People's Democratic Republic Nigeria Thailand 17 12 Turkey Viet Nam 29 61 19 100 46 32 98 43 115 79 65 42 328 200 Total number of cases includes number of deaths. WHO reports only laboratory-confirmed cases. http://www.who.int/csr/disease/avian_influenza/country/cases_table_2007_09_10/en/print.html

Seasonality of avian H5N1 viruses from domestic poultry in mainland in China Li et al., 2004 Nature 430, 209–213.

The genotypes of H5N1 reassortants(1999-2001) Guan et al., 2002 Proc Natl Acad Sci U S A 99, 8950–8955.

The genotypes of H5N1 influenza reassortants Li et al., 2004 Nature 430, 209–213

Phylogenetic relationships of the (A) HA and (B) NP genes of influenza A viruses isolated in Indonesia and Vietnam. Smith GJ, et al. Virology. 2006. 5;350(2):258-68.

Definition of avian influenza Intravenous pathogenicity index (IVPI) in 4-8 week-old chickens greater than 1.2, or cause more than 75% mortality within 10 days or nucleotide sequencing of H5 or H7 has demonstrated the presence of multiple basic amino acids at the cleavage site of the haemagglutinin. New proposed definition: IVPI in 6-week-old chickens greater than 1.2 or any influenza A virus of H5 or H7 subtype.

Birds are examined at 24-hour intervals for 10 days Birds are examined at 24-hour intervals for 10 days. At each observation, each bird is scored 0 if normal, 1 if sick, 2 if severely sick, 3 if dead. (The judgement of sick and severely sick birds is a subjective clinical assessment. Normally, ‘sick' birds would show one of the following signs and ‘severely sick' more than one of the following signs: respiratory involvement, depression, diarrhoea, cyanosis of the exposed skin or wattles, oedema of the face and/or head, nervous signs. Dead individuals must be scored as 3 at each of the remaining daily observations after death [when birds are too sick to eat or drink, they should be killed humanely and scored as dead at the next observation].) The intravenous pathogenicity index (IVPI) is the mean score per bird per observation over the 10-day period. An index of 3.00 means that all birds died within 24 hours, and an index of 0.00 means that no bird showed any clinical sign during the 10-day observation period. Office International des Epizooties (OIE)

Host protease-dependent activation of influenza virus NH2 NH2 HA１ S S Virus Activating Protease HA S S HA２ COOH COOH From Lance Jennings

Ubiquitous Protease present in Golgi Body Specific Secretory Protease Avian Influenza Viruses Ubiquitous Protease present in Golgi Body Furin Plasmin Fowl Plague Fatal Systemic Infection All tissues and organs HPAI Specific Secretory Protease Trypsin Tryptase Clara FXa Subclinical Localized Infection Respiratory & Alimentary Tractｓ ‘Epithelial cells’ LPAI Responsible Proteases Structure of HA Diseases in Chickens Tissue Tropism Virus HA1 HA2 - - - - - - R / - - - R R R R E K R / - - - Viral factors determining pathogenicity of avian influenza viruses 1. HA cleavage site Single or multiple basic amino acids: Susceptible to different host proteases 2. PB2 (polymerase) gene Amino acid change (E627 > K) among A/HK/97(H5N1) viruses --- mouse pathogenicity 3. NS1 gene (type 1 IFNs and TNF-a antagonism) Amino acid change (F92 > E or D?) --- Pathogenicity in pigs LPAI to HPAI in chickens? Passages of a LPAI H7 virus in CEC resulted in a multi-basic cleavage motif in HA 1) Insertion of a gene fragment of host cellular 28s r-RNA into the cleavage site 2) Insertion of a fragment of the homologous NP gene into the cleavage site and/or a few point mutations near the cleavage site A similar change was documented during natural transmission of a LPAI virus among chickens in Chile R J Webby, et al. 2004

NS gene of Influenza A 27 57 530 718 864 NS1 NS2

Influenza A virus NS1 protein RNA-binding domain (1-73) Effector domain (73-237) 1 19 34 36 38 73 137 146 186 216 221 223 237 RNA-binding domain (19-38) Nuclear localization signal (34-38, 216-221) Nuclear export signal (137-146) 30 kDa subunit of Cleavage and polyadenylation specific factor (CPSF) binding site (186) poly-A binding protein II (PABII) binding domain (223-237) PDZ domain ligand (228-231)

Virulence & cytokine response Importance of NS1 in Influenza A virus A D92E mutation in NS1 strongly affect the virulence of influenza virus, eg: the H5N1 avian flu virus (Seo et al. 2004) . C-terminal PDZ domain ligand in NS1 act as a potential virulence determinant (Krug et al. 2006) .

NS1 gene E92 and del 80-84 may affect RNA binding affinity H5N1 of Hong Kong outbreak Nat.Struct.Mol.Biol. 2006 E92 and del 80-84 may affect RNA binding affinity Cytokine-resistance

PDZ ligand motif in NS1 as a potential virulence determinant Large-scale sequence analysis of avian influenza isolates Obenauer et al. science. 2006 1997 PDZ domain Regulating the activity and trafficking of membrane proteins Maintaining cell polarity and morphology Organizing postsynaptic density in neuronal cells 2003 1918

Role of PB2 genes A E627K mutation in PB2 strongly affect the virulence of influenza virus, eg: the 1918 flu virus and the H5N1 avian flu virus (Gillis et al. 2005).

PB2 gene

Schematic diagram of chimeric and single amino acid PB2 mutants, with their virulence in mice (MLD50) mutation at position 627 in the PB2 protein influenced the outcome of infection in mice Hatta M, et al. Science. 2001. 7;293(5536):1840-2.

Mechanisms of pathogenesis of human H5N1 disease Malik Peiris, et al. Clin Micro Rev 20:243, 2007

Generation of pandemic influenza virus strains In this figure, let’s summarize the models for the generation of pandemic influenza virus strains. In the classical genetic reassortment model (A), avian and human viruses bind respectively to NeuAca2,3Gal and NeuAca2,6Gal (a2,3 and a2,6) linkages in the pig trachea, setting the stage for the emergence of a reassortant that infects lots of the human population. The segments in the center of each particle represent the viral genome. The black HA gene comes from an avian virus. In the adaptation model (B), avian viruses acquire the ability to replicate efficiently in humans during adaptation to the NeuAca2,6Gal linkage in pigs. This change is mediated by a mutation in the HA gene. Third, an avian influenza virus is transmitted directly to humans, where it reassorts with a human virus (C) or acquires the ability to recognize the NeuAca2,6Gal linkage after direct introduction from birds (D), leading to efficient replication in humans. Nichol, et al. PNAS 2000

Development of a vaccine Grow in eggs Grow in MDCK cells H5N1 is so deadly in chicken embryos, a new technique, known as “reverse genetics”, is required to prepare the prototype H5N1 virus for vaccine production. The virus can also be genetically modified so that it is no longer lethal to chicken embryos.

Vaccine of high pathogenic avian influenza virus Current vaccines are produced from virus grown in fertile hens’ eggs and inactivated by either formaldehyde or B-propiolactone. They consist of whole virus, detergent-treated split product, or purified haemagglutinin and neuraminidase surface antigen formulations of the three virus strains currently recommended by WHO. About 50 countries have government-funded national immunisation programs. However, when trying to make vaccine of H5N1 influenza virus A, the virus strain was too virulent for eggs to survive and replicate in them. In order to decrease its virulence, a scientist remove the additional basic amino acid of high pathogenic virus H5, then other protein genome was came from H1N1 virus strain. This vaccine was in clinical trial, but the efficiency of the recombinant virus vaccine still need to be determinated. Hoffmann, et al. 2000