papers in science and nature

The papers in Science and Nature

The newswires have been buzzing about the papers just outin Science

and Nature concerning the location of H5N1 attachment in the human

respiratory system. Most stories jump to the punchline: what it might

mean for human susceptibility and transmissibility. That part,

however, is interpretation and speculation. Let's look to see what

these papers actually say.

The central issue is the question of host range (or host specificity),

that is, what makes one influenza virus primarily a bird virus and

another one a human virus? This also bears on the ease of bird to

human transmission and possibly human to human transmission, although

the latter is where the speculation rather than the scientific results

enter. First a bit of background. If you want even more, we did a four

part series not so long ago that lays the science out in even more

detail here, here, here and here. I'll crib a little from them to save

time (after all, I'm just plagiarizing myself).

The influenza (or any) virus needs to get inside a host cell in order

to make new copies of itself. Reproducing is essentially its only task

in life. We know that viruses and other pathogens don't usually infect

all animals (they have a specific host range) and within an animal,

usually infect only specific tissues. So cells from different animals

and different tissues must somehow look different to the virus. How

does a virus "recognize" the right cell?

The first view the virus gets of the cell is a surface covered by a

dense canopy of sugars linked to cell surface proteins. This outer

fur-like sugar surface is called the glycocalyx and plays an important

biological role, including cell-cell recognition and communication,

interacting with and binding of cells to the material that glues cells

together (the extracellular matrix), altering or modulating the

response of immune cells and proteins, and, most important for our

purpose, protecting against or determining sensitivity to pathogens

like the flu virus. The influenza virus has learned to recognize one

of these projecting sugars and uses it to grab onto the cell and

initiate the process of getting inside it.

The particular sugar we are interested in is called sialic acid. It

often rests at the tips of a sugar chain in turn attached to proteins

that are part of the cell surface (see the earlier posts for pictures

and a lot more explanation). How the sialic acid is attached to the

other sugars in the chain is the key to what the papers are about. For

our purposes there are two ways this attachment can be done,

designated either an a-2, 3 or an a-2, 6 linkage. These denote two

different ways to attach the sialic acid tip to another sugar,

galactose, which is next in line in the sugar chain. Other sugars and

configurations further in may also be important but the papers at

issue consider only the Sialic Acid a-2, 3 or a-2, 6 Galactose

linkages (often written NeuAc a-2, 3 Gal or NeuAc a-2, 6 Gal because

another name for sialic acid is neuraminic acid; the two papers use

the designations SAa-2, 3Gal and SAa-2, 6Gal). This all may seem

somewhat esoteric, but it turns out that avian viruses like to bind to

sialic acid linked a-2, 3 while human viruses like the ones linked

a-2, 6.

The story used to be fairly simple. Birds had cells with SAa-2, 3Gal

visible and available for binding to the virus in their intestines and

influenza in birds is primarily an intestinal infection. Human beings

have SAa-2, 6Gal in their respiratory tract and the viral subtypes

that looked for that linkage were the ones that infected humans. Alas,

like everything else related to influenza, the story is more

complicated. For one thing it was discovered several years ago that

humans also have SAa-2, 3Gal on some of their cells. But which cells?

That's where these papers come in.

In 2004 Matrosovich and colleagues used a tissue culture system

derived from the human respiratory tract to try to figure out which

cells had a-2, 3 and which ones had a-2, 6 (Proc. Nat. Acad. Sci.

101:4620 - 4624. 2004). Their conclusion was that the avian virus

infected ciliated cells of the respiratory tract while the human

viruses infected the non-ciliated ones. The ciliated cells are higher

up and in the upper, mid and a bit of the lower respiratory tract,

decreasing in density as you go deeper. There are non-ciliated cells

throughout, too, but deep in the lungs, especially in the areas where

the gas exchange is taking place (the alveoli) the cells are

non-ciliated. The number of a-2, 6 cells in humans seemed to be

higher, but there were significant numbers of a-2, 3 throughout as

well. This was a bit of a puzzle. Why didn't the avian virus infect

humans more easily?After all, there were sufficient numbers of a-2, 3

cells in humans, too. Moreover, in human influenza infections,

ciliated cells throughout the upper respiratory tract are infected.

The story was unclear.

Matrosovich didn't use an intact human respiratory tract but a tissue

culture model of one, that is, one that had the same kinds of cells as

the respiratory tract but not organized into actual respiratory tract

tissue. The two new papers were designed to answer the question of

exactly which cells in the intact human respiratory tract the H5N1

virus preferentially bound.

The Dutch group didn't bother with determining a-2, 3 or a-2, 6

characteristics but instead incubated inactivated virus with a label

on it with archived formaldehyde preserved tissue sections and then

looked to see what part of the lung and which cells bound the virus.

The results were that H5N1 mainly bound to a type of cell in the

deepest part of the lung where gas exchange takes place (see the

earlier posts for an explanation of lung structure). The cell is

called a type II pneumocyte and it secretes a protein that helps the

lung stay expanded by decreasing its surface tension. Secretory cells

make abundant protein, which may be an advantage to the virus because

it hijacks the cell's protein making machinery to make copies of

itself. The virus also bound to wandering immune system cells called

pulmonary alveolar macrophages, which play an important part in

fighting pathogens and other garbage in the delicate tissues of the

deep lung where gas exchange is the main order of business.

The Japanese group (Nature) did look for a-2, 3 cells and found them

in the same place that the Dutch group saw viral binding: deep in the

lungs in the type II pneumocytes. Additionally they showed that avian

viruses bound to those human cells, as would be expected because they

had a-2, 3 sialic acid linkages. Cells higher in the human respiratory

tract (nose, throat, bronchial tubes that lead down into the lung) had

abundant a-2, 6 linkages. Human viruses tended to bind to and infect

cells higher in the respiratory tract, although some type II

pneumocytes also had a-2, 6 linkages.

These findings show virus infecting non-ciliated cells just as in

Matrosovich's study, but don't mention ciliated ones. So the story

isn't complete. Nor does it explain a number of things, for example,

if there are a-2, 3 and a-2, 6 studded cells throughout the human

respiratory tract, why do the avian viruses seem only to prefer the

ones down deep and the a-2, 6 viruses the ones further up? There are

quantitative differences in the density of these cells but the details

aren't known at the moment and other factors besides the sialic acid

linkage might be involved. The finding that H5N1 (an avian a-2, 3

virus) destroys the deep part of the lung has been found clinically

and is an explanation for the remarkably virulent nature of this

disease. Orange over at the excellent bird flu site The Coming

Influenza Pandemic? has resurrected several earlier stories

identifying type II pneumocytes as the site of the primary lesion in

fatal cases of bird flu, so this isn't a completely new finding.

Finally we come to the question of what it means. My answer may be

disappointing. At this point we don't know. The investigators

speculate (in the news stories more than the papers themselves) that

the reason bird flu is not as "catchable" as ordinary flu is that its

residence deep in the lungs makes its transmission more difficult.

There is no mucus in the gas exchange units so coughing and sneezing

is less likely to create a virus-containing aerosol. Or so it would

seem. In truth, however, we don't know the main routes of

transmission. Gas from the alveoli (the deep air sacs) is certainly

expelled on exhalation, likely contains virus, and once outside the

humidified environs of the respiratory tract would rapidly dessicate

(dry up) and could form droplet micronuclei. The assumption that virus

deep in the lungs is less transmissible might be correct but it has

not been shown. Other factors might be involved. Neither paper tested

the transmissibility question, which remains pure speculation

(although not implausible). The Japanese paper also points out that if

the virus were to develop the ability to dock with a-2, 6 cells,

either in addition to or instead of a-2, 3, we could have a nasty

actor on our hands. One isolate from Hong Kong in 2003 seems to have

this ambidextrous character, although most H5N1s do not.

That's my initial read of these two fascinating papers. They are a

step forward but leave much to be discovered. Contrary to the more

optimistic interpretations, it is too early to conclude that H5N1 is

not likely to be easily transmissible from person to person soley on

account of its location deep in the lungs. The papers do not show this