OIE National Trainers’ Course on HPAI Surveillance for Field Veterinarians and Village Animal Health Workers in collaboration with FAO

(Jakarta, Indonesia, 29-31 October 2007)

Mat YamageOIE Tokyo 　

Disease Profile: Disease Profile: Host-Pathogen Interactions Host-Pathogen Interactions

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 protei

n ）

(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

1 2341 PB2 Transcriptase: cap binding

2 2341 PB1 Transcriptase: elongation

3 2233 PA Transcriptase: protease activity (?)

4 1778 HA Haemagglutinin

5 1565 NPNucleoprotein: RNA binding; part of transcriptase complex; nuclear/cytoplasmic transport of vRNA

6 1413 NA Neuraminidase: release of virus

7 1027M1 Matrix protein: major component of virion

M2 Integral membrane protein - ion channel

8 890NS1

Non-structural: nucleus; effects on cellular RNA transport, splicing, translation. Anti-interferon protein.

NS2 Non-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 hours @ 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

H1H2H3H4H5H6H7H8H9H10H11H12H13H14H15,H16

N1 N2N3N4N5N6N7N8N9

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:HA226leu　

Avian　viruses:HA226gln

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 10-12 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

Avian H3 Human H2

Human H3

• 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

Merci

World Organisation for Animal HealthWorld Organisation for Animal Health

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