Avian flu H5N1 A bird adapted strain of Influenza A H5N1 is endemic in many areas of southeast Asia, and a highly pathogenic strain is now spreading globally. Nomenclature: Highly pathogenic avian influenza virus of type A of subtype H5N1 = HPAI A(H5N1) In 2013 the WHO confirmed 630 human cases that have killed 375 people since There is no sustained human-to-human transmission, but there is some evidence of limited human-to-human transmission of the virus Although it is often confused with other flu-like illnesses, especially the common cold, the flu is a more severe disease caused by a different type of virus. Several H5N1 vaccines have been developed and approved, and stockpiled by a number of countries, including the US, Britain, France, Canada, and Australia. Research has shown that a highly contagious strain that might allow airborne transmission between mammals can be reached in only a few mutations, raising concerns about a pandemic and bioterrorism.

Global distribution of highly pathogenic H5N1

Low pathogenic avian influenza H5N1 Low pathogenic avian influenza H5N1 (LPAI H5N1) also called "North American" H5N1 commonly occurs in wild birds. In most cases, it causes minor sickness or no noticeable signs of disease in birds. It is not known to affect humans at all. The only concern about it is that it is possible for it to be transmitted to poultry and in poultry mutate into a highly pathogenic strain.

Signs and symptoms The avian influenza hemagglutinin binds different sialic acid receptors compared to seasonal flue, causing it to replicate in the lower respiratory tract, and consequently leading to viral pneumonia. There is as yet no human form of H5N1, so all humans who have caught it so far have caught avian H5N1. No one knows if the symptoms of a humanized H5N1 flu will be consistent with those for a typical humanized influenza A virus. The reported mortality rate of highly pathogenic H5N1 avian influenza in a human is high; WHO data indicate 60% of cases classified as H5N1 resulted in death. However, there is some evidence the actual mortality rate of avian flu could be much lower, as there may be many people with milder symptoms who do not seek treatment and are not counted.

The different sites of infection of seasonal H1N1 versus avian H5N1 influences their lethality and ability to spread

Cytokine storm There have been studies of the levels of cytokines in humans infected by A(H5N1). Of particular concern is elevated levels of tumor necrosis factor-alpha, a protein associated with tissue destruction at sites of infection and increased production of other cytokines. The inflammatory cascade triggered by H5N1 has been called a 'cytokine storm' by some, because of what seems to be a positive feedback process of damage to the body resulting from immune system stimulation. Cytokines are a broad and loose category of small proteins that are important in cell signaling - they are released by cells and affect the behavior of other cells, and sometimes the releasing cell itself. A cytokine storm, also known as cytokine cascade and hypercytokinemia is a potentially fatal immune reaction consisting of a positive feedback loop between cytokines and immune cells, with highly elevated levels of various cytokines H5N1 induces higher levels of cytokines than the more common flu virus types.

Mutation rate and species selectivity Influenza viruses have a relatively high mutation rate that is characteristic of RNA viruses. The segmentation of its genome facilitates genetic recombination by segment reassortment in hosts infected with two different influenza viruses at the same time. A previously uncontagious strain may then be able to pass between humans, one of several possible paths to a pandemic. (Note: Reassortment is the mixing of the genetic material of a species into new combinations in different individuals.) The ability of various influenza strains to show species-selectivity is largely due to variation in the hemagglutinin genes. Hemagglutinin mutations can significantly alter the ability of viral hemagglutinin proteins to bind to receptors on the surface of host cells. Such mutations in avian H5N1 viruses can change it from being inefficient to efficient at infecting human cells. This doesn't mean that one amino acid substitution can cause a pandemic, but it does mean that one amino acid substitution can cause an avian flu virus that is not pathogenic in humans to become pathogenic in humans.

Reassortment can result in novel and highly pathogenic strains of human flu The viruses that caused the major pandemics of the past century emerged upon reassortment (that is, genetic mixing) of animal and human viruses.

H5N1 transmission studies in ferrets Several studies have shown that reassortment of seasonal H1N1 and A(H5N1) do not lead to transmissible viruses between ferrets. (Ferrets have been used as animal models for influenza since 1933.) Novel, contagious strains of H5N1 were created by Fouchier et al. Three mutations were introduced into H5N, and it was then passed from the noses of infected ferrets to the noses of uninfected ones, which was repeated 10 times. After, the H5N1 virus had acquired the ability of transmission between ferrets via aerosols or respiratory droplets. After Fouchier offered an article describing this work to the journal Science, the US National Science Advisory Board for Biosecurity (NSABB) recommended against publication of the full details, and the one submitted to Nature by Kawaoka et al. describing related work. However, after additional consultations at the WHO and by the NSABB, they reversed their position and recommended publication of revised versions. However, the Dutch government then declared Fouchier had to apply for an export permit in the light an EU ‘dual use’ directive. After much controversy, Fouchier complied (under formal protest) with demands to obtain a special permit for submitting his manuscript, and his research appeared in a special issue of the journal Science devoted to H5N1. Both papers conclude that it is entirely possible that a natural chain of mutations could lead to an H5N1 virus acquiring the capability of airborne transmission between mammals, and that a H5N1 influenza pandemic would not be impossible.

Serial passage Serial passage is a virus attenuation technique developed originally by Louis Pasteur in the 1880s. It is similar to selective breeding, and can be used to create an attenuated strain of a virus to develop vaccines, or to increase the virulence of a viral strain in order to create epidemics. The process involves infecting a series of host organisms with a virus. Each time the virus is given some time to incubate, and then the next host is infected with the incubated virus. The virus may mutate repeatedly into a form that is resistant to a wide variety of host immune system defenses, or a weaker strain may result. Louis Pasteur

Approach

Key results Conventional Sanger sequencing was used to determine the consensus genome sequences of viruses recovered from the 6ferrets that acquired virus via airborne transmission. All six samples still harbored substitutions Q222L, G224S, and E627K that had been introduced by reverse genetics. Surprisingly, only two additional amino acid substitutions, both in HA, were consistently detected in all airborne- transmissible viruses: (i) H103Y, which forms part of the HA trimer interface, and (ii) T156A, which is near the HA receptor binding site. Although we observed several other mutations, their occurrence was not consistent among the airborne viruses, indicating that of the heterogeneous virus populations generated by passaging in ferrets, viruses with different genotypes were transmissible. Together, these results suggest that as few as five amino acid substitutions (four in HA and one in PB2) may be sufficient to confer airborne transmission of HPAI A/H5N1 virus between mammals. None of the six ferrets died from the virus.

Hemagglutinin mutations

Polymerase Basic Protein 2 Polymerase Basic Protein 2 (aka PB2) is a protein subunit that together with Polymerase Basic Protein 1 and Polymerase Acidic Protein forms an RNA dependent RNA polymerase.

The transmissible viruses were sensitive to Tamiflu

Abstract Highly pathogenic avian influenza A/H5N1 virus can cause morbidity and mortality in humans but thus far has not acquired the ability to be transmitted by aerosol or respiratory droplet (“airborne transmission”) between humans. To address the concern that the virus could acquire this ability under natural conditions, we genetically modified A/H5N1 virus by site-directed mutagenesis and subsequent serial passage in ferrets. The genetically modified A/H5N1 virus acquired mutations during passage in ferrets, ultimately becoming airborne transmissible in ferrets. None of the recipient ferrets died after airborne infection with the mutant A/H5N1 viruses. Four amino acid substitutions in the host receptor-binding protein hemagglutinin, and one in the polymerase complex protein basic polymerase 2, were consistently present in airborne-transmitted viruses. The transmissible viruses were sensitive to the antiviral drug oseltamivir and reacted well with antisera raised against H5 influenza vaccine strains. Thus, avian A/H5N1 influenza viruses can acquire the capacity for airborne transmission between mammals without recombination in an intermediate host and therefore constitute a risk for human pandemic influenza.

Approach Generate hybrid virus with H5N1 HA and pandemic H1N1 remaining 7 segments Generate library of HA mutants Identify mutants likely to lead to ferret-to- ferret transmission by selecting for mutants that bound to mammalian-like receptors that had been added to the surface of turkey red blood cells In vivo evaluation of the best hits

Hemagglutinin mutations a, Close-up view of the globular head of VN1203 HA. Mutations known to increase affinity to human- type receptors are shown in blue. Amino acid changes not previously known to affect receptor binding are shown in green. Additional mutations that occurred in the HA of H5 avian–human reassortant viruses during replication and/or transmission in ferrets are shown in red. b, The positions of four mutations in the HA of H5 transmissible reassortant mutant virus, HA(N158D/N224K/Q226L/T318I)/CA04, are highlighted in red. The fusion peptide of HA is shown in cyan.

Abstract Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host-range determinant as it mediates virus binding to host-specific cellular receptors. Here we assess the molecular changes in HA that would allow a virus possessing subtype H5 HA to be transmissible among mammals. We identified a reassortant H5 HA/H1N1 virus—comprising H5 HA (from an H5N1 virus) with four mutations and the remaining seven gene segments from a 2009 pandemic H1N1 virus— that was capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but was not highly pathogenic and did not cause mortality. These results indicate that H5 HA can convert to an HA that supports efficient viral transmission in mammals; however, we do not know whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral gene segments may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian–human reassortant viruses as tested here, may emerge. Our findings emphasize the need to prepare for potential pandemics caused by influenza viruses possessing H5 HA, and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production and distribution of effective countermeasures.

Conclusions Herfst et al.HA: H103Y, T156A, Q222L, G224S PB2: E627K Imai et al.HA: N154D, N220K, Q222L, T314I (note: numbers are shifted to Herfst numbering scheme) In summary… Hemagglutinin is key. Transmission can be conferred in a relatively straightforward way with a minimal number of mutations. Mutations tended to cluster around sites of known importance. Ferrets got sick and infected other ferrets; however, ferret-to-ferret illnesses never lead to death.

Questions Should this work have been performed? Should it have been published? Do you think what was held back was sufficient?