Mapping Avian Flu Mutations

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Mapping Avian Flu Mutations

Scientists are using a variety of tools and techniques to determine

how the H5N1 influenza virus is mutating.

By Shaffer

The spread of influenza A subtype H5N1 beyond its usual reservoirs in

Asia has triggered a worldwide flu epidemic—for birds. Since

historical flu pandemics are thought to derive from avian viruses,

the race is on to learn as much as possible about the molecular

determinants of virulence and pathogenicity in the virus and prevent

a new pandemic. Scientists trying to track potential mutation

pathways for H5N1 have made a number of key findings, including one

that calls into question whether a single mutation in H5N1 could lead

to a human pandemic. It could turn out that avian influenza is

literally for the birds.

The flu virus is a remarkably simple organism, being an RNA virus

composed of only eight genes. The two most well known genes are for

hemagglutinin (HA) and neuraminidase (NA). The NP gene encodes a

nucleoprotein, the M gene encodes two different matrix proteins in

different reading frames, and the NS gene encodes non structural

proteins NS1 and NS2, also using different reading frames. The three

remaining genes, PA, PB1, and PB2, encode the three RNA strands that

make up the RNA polymerase.

The flu life cycle begins when the viral envelope, with its two

surface glycoproteins, HA and NA, binds to a sialic acid-terminated

oligosaccharide on the surface of the host cell via HA. The virus

enters the cell through endocytosis. The host cell machinery

transports the genetic material to the nucleus where transcription

and replication begin. Because viral replication happens rapidly, the

cell is quickly overwhelmed and usually dies as a result of the

infection. At the end of the cycle, the viral particles are assembled

and the surface proteins HA and NA once again mediate transport

through the cell membrane, this time from the inside to the outside.

Surface specificity

Donis, PhD, is chief of the Molecular Virology Branch of the

Influenza Division at the Centers for Disease Control (CDC), Atlanta.

One of their primary goals with H5N1 is identifying markers of

virulence and pathogenicity to be able to perform receptor-binding

studies, cellular analysis, and sequence analysis on various viral

strains. This would allow the CDC to assess the danger of H5N1

samples without relying on animal manipulation studies using mice and

ferrets.

Donis and colleagues extract RNA from viruses grown in chicken eggs

using off-the-shelf RNA extraction and amplify it using reverse

transcription polymerase chain reaction (PCR). The resulting DNA

sequence can then be analyzed and aligned with other viruses,

yielding valuable information about phylogenetic relationships of the

virus, particularly the highly variable HA gene.

The researchers created a phylogenetic map for the viruses, based on

sequence similarities in the HA gene, and divided the virus strains

into clades. It was found that strains of virus isolated from human

beings, most of who succumbed to the disease, were highly pathogenic

in ferrets, whereas strains of H5N1 isolated from chickens were not

highly pathogenic in ferrets.

Donis and his group continue to closely monitor changes in the HA

binding pocket that would indicate the virus is acquiring the ability

to recognize human receptors. The sialic acid linkage on the host

cell surface is crucial. Avian-adapted flu viruses preferentially

bind to sialic acids that are linked in an á-2,3 configuration with

galactose. This is the sugar that is most abundant in the respiratory

tract of chickens and ducks. In well-adapted human flu viruses, such

as seasonal H1-type influenza, the HA is optimized to bind to an á-

2,6-linked sialic acid. The á-2,6 linkage has a bend or a kink in it,

compared to the stick-like á-2,3. One of the important differences

between avian- and human-adapted influenza viruses is that the two

types of HA have different amino acids lining the binding pocket to

either accommodate the straight-line linkage or the kinked linkage,

respectively.

Donis says there are two amino acid positions that could determine

this specific interaction. " We have very good knowledge of what

happens in the transmission from ducks to humans with previous

pandemic virus. The H3 virus that came into the human population

in `68 had changes at positions 226 and 228. The virus that came into

humans in 1918, the so-called Spanish flu, is a different subtype,

H1. That one had changes at 190 and 225. "

The poor ability of H5N1 to bind to cell receptors on human host

cells may be mechanistically responsible for the different clinical

course of the infection from human-adapted flu virus subtypes. " The

current thinking is that of all the people that are exposed, very,

very few get sick, " Donis says. " If you look at the people that are

getting sick, the virus is located not in the upper respiratory

tract, but in the alveoli, at the very end of the lungs . . . What

happens is that the virus has to get deep, deep, deep to gain a

foothold and replicate. Fortunately for us . . . the return trip is

equally difficult. "

This means that even though the infected individual may become very

ill, he or she is unlikely to transmit the infection to other people.

This raises the question of whether an adaptation of the HA gene to

accommodate the human conformation a-2,6 receptor would at the same

time reduce the massive and deadly infection to something resembling

the seasonal flu. " If the virus has to gain the ability to infect the

upper respiratory tract, it would lose virulence. If it gains the

ability to infect the upper respiratory tract, while retaining the

ability to infect pneumocytes, we could have a very nasty virus. "

One theoretical bridge between avian and human flu is the pig. Swine

have both á-2,3 and á-2,6 receptors on their cells, and thus are

highly susceptible to both avian and human viruses. The theory is

that the pig can serve as a host in which the genes of the two

different viruses can shuffle and recombine, resulting in a new

strain of virus.

Core proteins and beyond

At St. Jude Children's Research Hospital in Memphis, Tenn., some of

the world's leading influenza experts are working to characterize the

pathogenic potential of recently emerging H5N1 for mammalian species.

Their studies have revealed candidates in viral proteins other than

HA and NA. In a paper published on January 26 in Sciencexpress, 15

authors, including St. Jude's Webster, PhD, and h

Hoffmann, PhD, report the results of a massive gene sequencing

project, the first of its kind for avian influenza.

Currently, public repositories of genetic information for influenza

are heavily skewed toward surface glycoproteins and shorter genes

such as M and NS. In order to identify species-specific markers of

pathogenicity, the group sequenced 4,339 genes from hundreds of

different avian influenza strains. In addition to grouping the virus

genomes into clades (a standard approach for phylogenetic analysis),

the group also created proteotypes for them based on unique amino

acid signatures.

From this analysis, the viral protein NS1 emerged as a potential

virulence determinant. NS1's exact function is not known, but it may

play a role in mediating cell defenses, such as interferon. This new

analysis revealed that NS1 contained a previously unknown PDZ domain

ligand. Since PDZ domain proteins are important in many structural

and signaling pathways in the cell, it is possible that avian NS1

widely disrupts these cellular activities.

With NS1 on the table as an important determinant of species

specificity in influenza virus, it is clear that the search cannot be

limited to HA and NA. Hoffmann studied a number of other viral genes

to see if there was an effect on virulence. He and his colleagues

amplified the viral genes using real time PCR with a standard set of

primers for influenza A virus, then inserted the viral cDNAs into

plasmids. After confirming that the virus regenerated from MDCK/293 T

cells was identical to the parent viruses, the team set about re-

assorting the genes by combining the desired plasmids. Initial

experiments exchanging the HA and NA genes in one of the same highly

pathogenic strains of H5N1 analyzed by Donis et al with a less

pathogenic chicken strain revealed that HA and NA were not sufficient

to make the chicken strain highly pathogenic. " We swapped H and N,

and they didn't have an effect. They didn't determine the

virulence...hemagglutinin is not sufficient, " Hoffmann says.

When Hoffmann switched out all three polymerase genes from the

harmless chicken virus into the lethal human strain virus, all

animals survived, demonstrating that the polymerase genes are

important for virulence. The reverse experiment resulted in a chicken

virus with three polymerase genes from the human-derived virus which

was lethal to mice, but which ferrets survived after marked symptoms.

The three polymerase genes, PA, PB1, and PB2,would seem to be

important in the virulence of the virus and linked because single-

gene swaps did not result in dramatic changes in virulence.

The interrelation of these three genes suggests that changes or

mutations affecting one gene may require a complementary change in

one of its partners. On a larger scale, the analysis of the St. Jude

team revealed that the vast majority of complete genomes in their

sample shared at least five genes with one other genome. This means

that mutations of highly conserved genes seem to be complemented by

changes in other genes at the same time. This calls into question any

suggestion that a single mutation in H5N1 could lead to the feared

human pandemic.

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