OIE National Trainers’ Course on HPAI Surveillance for Field Veterinarians and Village Animal Health Workers in collaboration with FAO (Jakarta, Indonesia, October 2007) Mat Yamage OIE Tokyo

At a poultry farm, somebody might ask you a question: “What are you doing?” -“I am doing what I was to told to do” - “I am earning living expenses” - “I am trying to prevent an outbreak of avian influenza”

(~200nm in diameter) Influenza virus A/duck/Hokkaido/5/77(H3N2 ) Electron micrograph of the Influenza virus Professor KIDA Hiroshi HPAI(highly pathogenic avian influenza) infected chicken （ Arrow points to hemagglutinin protein ）

(Nephelium lappaceum L.) Rambutan fruites (Castanea crenata L) Japanese Chestnut Iinfluenza viruses are spherical, covered with spiky protein projections like those on the surface of a chestnut conker shell. Morphology of the Avian Influenza virus reminds us a chesnuts or rambutan

Segment:Size(nt)Polypeptide(s) Function PB2Transcriptase: cap binding PB1Transcriptase: elongation PATranscriptase: protease activity (?) HAHaemagglutinin NP Nucleoprotein: RNA binding; part of transcriptase complex; nuclear/cytoplasmic transport of vRNA NA Neuraminidase : release of virus M1Matrix protein: major component of virion M2Integral membrane protein - ion channel NS1 Non-structural: nucleus; effects on cellular RNA transport, splicing, translation. Anti-interferon protein. NS2Non-structural: nucleus+cytoplasm, function unknown Eight segments of avian influenza virus genome

The outer surface of the particle consists of a lipid envelope from which project prominent glycoprotein spikes of two types: haemagglutinin (HA), a 135Å trimer neuraminidase (NA), a 60Å tetramer The inner side of the envelope is lined by the matrix protein. The particles are relative labile (half-life a few R.T.), not resistant to drying, etc.

Haemagglutinin. A simplified 3D model of influenza virus surface glycoprotein haemagglutinin. HA codes for haemagglutinin, an antigenic glycoprotein found on the surface of the influenza viruses and is responsible for binding the virus to the cell that is being infected.

NA codes for neuraminidase, an antigenic glycosylated enzyme found on the surface of the influenza viruses. It facilitates the release of progeny viruses from infected cells Neuraminidase

Viruses cannot reproduce on their own as they are dependent on host metabolic machinery and ribosomes to replicate and reproduce.

Entry into the host cell and nucleus Entry into the cell- facilitated by binding of the HA spikes to mucoproteins containing terminal N-acetyl neuraminic acid (NANA = sialic acid) groups present on the host cells. After adsorption – engulfed - internalized as an endosome. – acidified to be cleaved by trypsin-like enzymes into the HA1 and HA2 (transmembrane COOH portion) polypeptides (linked by disulphide bonds The combination of the close proximity of the virus envelope and the membrane of the endosome and the active membrane-fusion domain of HA2 results in fusion of the two membranes and passage of the nucleocapsid into the cytoplasm Cleaved HA spike activates the membrane-fusion function located in HA2. T

Different types of RNAs formed in the host nucleus by influenza viruses Transcription of the influenza virus genome segments : s/s (-)sense RNA in 8 segments are are transcribed by the 3 polymerase polypeptides associated with each genome segment. The 5' and 3' terminal sequences of all the genome segments are highly conserved: The genome segments are packaged into the core. The RNP (RNA + nucleoprotein, N) is in a helical form with the 3 polymerase polypeptides associated with each segment. © Paul Digard, Dept Pathology, University of Cambridge. \

Species Infected by Influenza A, HA and NA Subtypes H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15,H16 N1 N2 N3 N4 N5 N6 N7 N8 N9

Species barrier: The species which different types of influenza viruses are able to infect are determined by different forms of sialic acid present on the host tissue. In particular, this property depends predominately (but not exclusively) on the amino acid at position 226 of the haemagglutinin protein: Human viruses: HA226 leu Avian viruses: HA226 gln This provides a considerable species barrier between birds and humans which is not easily overcome. However, pigs provide a "mixing pot" - able to be infected by both types of virus & thus allowing the passage of avian viruses to humans.

Generally, Avian viruses do not infect humans, but they do have potential to cross the "species barrier" and develop into new viral strains that are infectious to humans. Several theories have been put forward to explain the origin of new strains. A few are as follows: How the host ranges are expanded

Antigenic Drift Lesser antigenic changes due to spontaneous genetic mutations over time, due to natural selection pressure of antibodies. For examle, RNA segment coding for H protein Though pandemics arise due to antigenic shifts every years, smaller epidemics can occur regularly in the intervening years. The viruses isolated from such epidemics showed strain differences when compared in the HAI tests, i.e. although the viruses belong to the same subtype, they do not crossreact completely.

Antigenic shift Reassortments new combinations of H and N can occur by reassortments of the RNA segments coding for H and N proteins when cells are coinfected with 2 different sub-type viruses people, pigs and aquatic birds are the principle variables associated with the interspecies transfer with pigs and ducks acting as “mixing vessels”

Predominance of the SA 2,3Gal linkage as detected by lectin staining in horse trachea. The MAA lectin specific for SA 2,3Gal ( 2-3; detected with fluorescein isothiocyanate (FITC)-labeled anti-DIG antibody) bound to horse and pig tracheal epithelium, whereas SNA lectin specific for SA 2,6Gal ( 2-6; detected with rhodamine-labeled anti-DIG antibody) bound only to the latter. Blue staining is a nonspecific reaction. Suzuki et al., 2000, JV 74:11825

Existence of both Sia 2-3Gal and Sia 2-6Gal linkages in quail and chicken colons detected by lectin staining. Both DIG-labeled MAA lectin specific for Sia 2- 3Gal and DIG-labeled SNA lectin specific for Sia 2-6Gal bound to quail and chicken colon epithelium, and then their binding specificities detected with polyclonal sheep anti-DIG Fab fragments conjugated with alkaline phosphatase

This theory is based on the view that the new virus subtypes are reassortant viruses resulting from dual infection. The 8 ssRNA segments of each strain reassort with each other, producing a new subtype. IA viruses can cross the "species barrier," and pigs are postulated as the most likely "mixing vessel." Pigs and birds are believed to be particularly important reservoirs, generating pools of genetically/antigenically diverse viruses which get transferred back to the human population via close contact between humans and animals.

Reassortment

Interactions between avian influenza viruses and hosts and resultant disease manifestations differ depending on the combination of the type of viruses and the host animal species. Host specificity, tissue tropism and pathogenicity could be determined by the genetic alteration in HA and NA of avian influenza viruses. A host or tissue with receptors for multiple types of avian influenza viruses could play a role of melting pot for genetic materials leading to expanding the host range and augment pathogenicity. SUMMARY

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