As these avian strains are normally highly pathogenic to birds, they must be attenuated in order to permit growth in eggs. This is achieved through the removal of a gene segment coding for a polybasic stretch of amino acids at the hemagglutinin cleavage site. The genetically modified hemagglutinin and neuraminidase from the H5N1 strain are then reassorted in cell culture by a process known as reverse genetics to construct the vaccine seed (see diagram below - click the diagram to see it in full size).

A flu virus contains eight gene segments. One of the gene segments codes for the surface antigen hemagglutinin (HA) and another codes for the surface antigen neuraminidase (NA).

Scientists can custom-make a flu strain by assembling genes that code for the desired features. Two genes representing the HA and NA antigens are selected from the target strain (flu strain 1), while the remaining six genes come from a virus that's time-tested for its ability to grow inside an egg (flu strain 2). (Although the influenza virus actually uses RNA as its genetic material, the researchers make complementary pieces of DNA because DNA is easier to work with.)

1. After removing the dangerous part of the HA gene, scientists splice the HA and NA genes from flu strain 1 into circular pieces of DNA called plasmids.
2. Additional plasmids are created using the remaining six genes found in flu strain 2.
3. Scientists insert the HA and NA plasmids from flu strain 1 and the six plasmids carrying genes from flu strain 2 into animal cells growing in the laboratory.
4. The genes in the plasmids instruct the animal cells to make the desired new flu strain

The use of reverse genetics has allowed avian strains such as H5N1 to be grown in eggs, also minimizing risk to the personnel working in the production facility.

The newly developed cell culture manufacturing facilities operate at higher biological safety levels than egg production facilities, because they are closed systems. This means that they are capable of using the unmodified wild type virus, without waiting for the development of seed virus, from reverse genetics or reassortment. This could result in production time savings and earlier vaccine availability at the beginning of a pandemic.