Since then, the world follows the circulation of this virus with concern. Although we associate it to the severe cases in humans, this type of Influenza is also able to cause heavy symptoms in birds. Therefore, it is classified into two types: highly pathogenic or HPAI, which causes thousands of deaths in wild and domestic birds, and severe health problems in humans, and the low pathogenic or LPAI that infect birds and is asymptomatic. …………………………………………………………………………………………………………………….

The highly pathogenic H5N1 cause several health complications in humans, with nervous and hepatic damages, in addition to damages to the respiratory tract. Despite this, it is not efficiently transmitted among humans, being contracted by those in direct contact with domestic birds or close persons.

Currently, it is known that H5N1 has been established and circulating in poultry birds in Asia. The virus had already been detected in several countries of Asia, Europe and Africa, being transferred by migratory birds and many times contaminating local domestic birds. [2]

The highly pathogenic H5N1 has a number of uncommon characteristics that concern us. The most obvious of them is the fatality; no other influenza is able to kill such a high number of the infected. More than half the persons that were confirmed to have contracted it died. Due to a number of complications that ranges from the most common such as respiratory problems to the most severe and rare such as neurological complications. From the 442 cases reported to the WHO up to now, 262 were fatal. It is important to emphasize the fatality must be less than that recorded, once not all go to hospitals, especially the asymptomatic cases.

It is not only the cases in humans that concern us. Livestock are also seriously affected. Farms where outbreaks occurred resulted in high losses by infection, without considering the cases where the animals are sacrificed to prevent the spread of the virus generally to all other places and surroundings. In the wild environment, undomesticated animals are often found dead and tested positive to the virus, such as eagles and geese.

Another reason for concern is the broad number of hosts that the H5N1 can infect. Besides a high diversity of birds (geese, swans, turkeys and even flamingos, among others) several animals that usually are not connected to the flu have been found with the virus, including large felines such as tigers and leopards that feed on chickens at a Zoo in Thailand. Even a cat was infected after eating a pigeon in 2004, also in Thailand. [3]

Up to now, only one case of transmission between humans was confirmed in 2005, and some are still suspects but without confirmation. One sick child transmitted the virus to his mother, certainly involving close contact between them. This scarcity of contamination events between people indicate a low efficiency of the virus in propagating among humans.

Even not being transmitted between humans, scientists, health agents and the government closely monitor the circulation of H5N1. In the next text, we will see the characteristics of the virus that can contribute to the prevention of its transmission and those that explain its pathogenicity, and what else that give us more reasons to worry about.

Sources:
[1] Beigel JH, Farrar J, Han AM, Hayden FG, Hyer R, de Jong MD, Lochindarat S, Nguyen TK, Nguyen TH, Tran TH, Nicoll A, Touch S, Yuen KY, & Writing Committee of the World Health Organization (WHO) Consultation on Human Influenza A/H5 (2005). Avian influenza A (H5N1) infection in humans. The New England journal of medicine, 353 (13), 1374-85 PMID: 16192482
[2] Chen, H. (2006). Establishment of multiple sublineages of H5N1 influenza virus in Asia: Implications for pandemic control Proceedings of the National Academy of Sciences, 103 (8), 2845-2850 DOI: 10.1073/pnas.0511120103
[3] H5N1 avian influenza: Timeline of major events. 27 July 2009. (pdf)

The influenza H7 comprises various lineages, H7N7, H7N3 and H7N2 are those who knowingly infect humans. Some lineages are not very pathogenic (LPAI) and others highly pathogenic (HPAI), the majority circulates in birds and several mammals, especially horses. In Canada in 2004 and in England in 2006, domestic bird breeders contracted the H7N3, and in 2007, the H7N2 also infected breeders. In all the cases, the flu had mild respiratory symptoms and conjunctivitis occurred in some victims. The conjunctivitis is not a common symptom in seasonal flu, which is contracted often.

However, the H7N7 caused more problems. In Holland in 2003, after the outbreak of H7N7 in domestic birds, 89 persons were confirmed having this virus. The respiratory symptoms were mild to moderate and again some developed conjunctivitis. One death by severe pneumonia and related symptoms was recorded. [1]

Other cases of H7N7 have already been recorded, all involving breeders. From ducks to a seal that sneezed on his caretaker, always with the concomitant occurrence of conjunctivitis. [2]

H9N2 is, however, restricted to birds, and its closest proximity with us is through poultry birds, especially ducks and turkeys. On March 1999, two children in Hong Kong were found with this virus and the symptoms were fever and irritation in the throat. Both cases were solved without complications some days later. Here, the pattern was recurrent, although the cases were in regions far from each other; in both situations the children reported having recent contact with poultry birds. [2]

In 2003 and later in 2007, this virus infected humans once again in Hong Kong. Again, two children. Both developed some flu symptoms and were cured. In the case of the 2003 virus, its genetic material showed the origin of the virus to be among poultry birds in the Hong Kong market. There are other recorded cases, all in the same region. [3]

Although the recorded cases of H9 and H7 are few, and with mild symptoms, these viruses are disturbing for being some of the few bird lineages transmitted to humans and between humans, with a potential to originate a highly pathogenic lineage directly or by the rearrangement with other types of Influenza A circulating in people.

A very disturbing characteristic of these cases is the frequency of contaminated breeders. After all, they are in direct contact with the animals, much more exposed than the majority of the people. In a recent study with over 2000 persons from various professions in China, 4.5% tested positive to anti H9 antibodies, indicating that they had been in contact with this lineage before. Among poultry bird sellers, the positive rate was much higher, at 15.5%. The response against H5 was also detected in 0.2% of the tested persons. [4]

This association with breeders and sellers shows the importance of monitoring the type of virus that can be contracted, as well as instructing them about the precautions to avoid infection and transmission to other people. In the case of H9N2, there is also the possibility of the production of vaccines for domestic animals, which should also help in protecting the owners.

Workers in constant contact with both domestic and wild animals can be an important link in the circulation and transmission of new lineages to humans.

Sources:

[1] DEWIT, E., & FOUCHIER, R. (2008). Emerging influenza Journal of Clinical Virology, 41 (1), 1-6 DOI: 10.1016/j.jcv.2007.10.017
[2] Subbarao*, K., & Katz, J. (2000). Avian influenza viruses infecting humans Cellular and Molecular Life Sciences, 57 (12), 1770-1784 DOI: 10.1007/PL00000657
[3] Butt, K., Smith, G., Chen, H., Zhang, L., Leung, Y., Xu, K., Lim, W., Webster, R., Yuen, K., Peiris, J., & Guan, Y. (2005). Human Infection with an Avian H9N2 Influenza A Virus in Hong Kong in 2003 Journal of Clinical Microbiology, 43 (11), 5760-5767 DOI: 10.1128/JCM.43.11.5760-5767.2005
[4] Wang M, Fu CX, & Zheng BJ (2009). Antibodies against H5 and H9 avian influenza among poultry workers in China. The New England journal of medicine, 360 (24), 2583-4 PMID: 19516044

In a broad way and with many exceptions, infectious diseases may be divided into acute and chronic. The acute infection occurs when the virus quickly infects the host, causes symptoms, is or is not transmitted and the disease is contained. It is the case of influenza, measles and dengue. In contrast, there are chronic infections such as hepatitis C, HIV and Tuberculosis, which cause long-term diseases and lighter symptoms at the first time.

Not rare, the virus that causes acute infection has a seasonal cycle, that is, with well marked waves of epidemics. The cycles can be regulated by various factors. Seasons of the year, immunity of the susceptible population, life cycle of the vector (as the case of dengue, which depends on the Aedes aegipty mosquito that reproduces during the raining season in order to be transmitted) are some of the influencing factors. [1]

Percentage of flu cases per week. Countries closer to the north and the south such as USA and Argentina have well concentrated cases. Source: reference 4.

In the case of Influenza, the seasonality is well characterized and latitude-dependent. Countries of temperate climate, closer to the North and the South have well defined flu seasons during winter, which corresponds to the months of December, January and February in the North and June, July and August in the south. Tropical countries, however, maintain a relatively constant number of cases during the year. The known possible reasons for this are many, but still far from being established.

We can see in the graph that countries in the more extreme latitudes, the incidence of cases is concentrated during some weeks of the year, the common flu season that lasts from 5 to 10 weeks. This is why the vaccination campaigns have to occur about one month in advance. Thus, there is enough time to produce immunity before the arrival of winter.

Mechanisms associated to the virus and host have been proposed to explain this cycle. From the human part, various agglomerations in closed environments during winter, coexistence of children at school and even fluctuations in our metabolism and immune response, such as decline of melatonin and vitamin D during winter have been considered possible explanations. [2]

More recently, studies focused on the properties of the virus raised promising results. In a recent reanalysis of the data presented by several articles, it was observed that the pressure of air vapor, that is, the absolute quantity of water dissolved in air, has a negative effect on Influenza. The greater the absolute humidity, the lesser is the transmission and the viability of virus in the air. – Relative humidity is the quantity of water dissolved in air in respect to how it behaves before precipitation (such as rain), the hotter it is, the more water can be dissolved; absolute humidity is the total quantity of water dissolved in air, regardless of how close it is to precipitating.

That is, during winter, the air is colder and there is less water dissolved in it. Therefore, the virus survives (continues to be infectious) in the air for a longer period of time and is transmitted more efficiently. During summer, the temperatures increase, more water is dissolved in the air and the virus is viable for a lesser period of time, making the cases of flu to be concentrated in winter. These results suggest that in closed places, such as daycare centers and schools, air humidifiers can be a good way of preventing the transmission of flu. [3]

The fact that the virus is best transmitted at a low vapor pressure can be related to the formation of aerosols and their remaining in the air for longer periods of time. Therefore, the doubt remains which would be the transmission way in countries with tropical climate that can maintain the virus in circulation along all the year.

The idea in this case is that, probably, in tropical countries, the transmission occurs especially through ways that involve contact, the contact with sick people and with contaminated surfaces, which came into contact with saliva or mucus containing the virus.

This will help explain the transmission of Influenza A (H1N1) in countries that had summer season from June to August. As the majority of the population do not have any prior immunity against this virus, it replicates more efficiently and also as a result of easier transmission by contact, once the conventional H1N1 and the H3N2 were not detected circulating during the same period of time. [4]

The seasonal cycles regulate the production of vaccines and the circulation of new variants of the virus, and are one of the main points still unanswered about the flu. Despite all the understanding that we have about the flu, one of the main points about the epidemics that still remains unknown and shows how much we still have to study and understand about the Influenza.

Sources:
[1] Leggiadro, R. (2001). Seasonal Variation of Host Susceptibility and Cycles of Certain Infectious Diseases The Pediatric Infectious Disease Journal, 20 (10) DOI: 10.1097/00006454-200110000-00027
[2] Lipsitch, M., & Viboud, C. (2009). Influenza seasonality: Lifting the fog Proceedings of the National Academy of Sciences, 106 (10), 3645-3646 DOI: 10.1073/pnas.0900933106
[3] Shaman, J., & Kohn, M. (2009). Absolute humidity modulates influenza survival, transmission, and seasonality Proceedings of the National Academy of Sciences, 106 (9), 3243-3248 DOI: 10.1073/pnas.0806852106
[4] Lowen, Anice; Palese, Peter. (2009). Transmission of influenza virus in temperate zones is predominantly by aerosol, in the tropics by contact: A hypothesis. PLoS Currents Influenza:RRN1002.

Our knowledge of Influenza in pig dates back to at least 1918 when it was observed that they could also catch the flu during a time when the human flu caused an uneven pandemic. In 1930 the virus was isolated in pigs, an H1N1 called the classic lineage, close to the human H1N1 and from the same origin: the H1N1 influenza from birds.

This virus was practically the only one circulating in pigs of North America until the end of the last century. It also circulated in pigs from Europe and Asia through a contamination in Italy in 1976, but in 1979 a new avian H1N1 completely substituted it. Since then, the history of the swine influenza and our influenza has been intercalating.

The recent world attention was more focused on the avian viruses, mostly due to the deadliness and fear caused by the H5N1, but pigs were also considered a possible source of pandemic viruses. There are several reasons that concern us about pigs, and the main ones are related to the swine physiology.

Pigs have both types of receptors for Influenza in the respiratory system, the sialic acid α2,3 and α2,6. While the virus circulating in birds have difficulties to infect us – because it uses mostly α2,3 and we only have this receptor in the lower respiratory tract (lung region), which makes the spread by cough or sneeze difficult – if this virus enters pigs it will find the α2,3 in the entire respiratory system, including the upper respiratory system. It will also find the α2,6 that, if it is able to use it, it will guarantee a higher chance of transmission among humans.

There is also the issue of temperature. Birds have a more active metabolism than ours, chickens for instance have an average temperature of 42ºC, in such a way that a virus adapted to replicate in birds generally has its enzyme functioning with less efficiency in humans. Pigs, however, have an average temperature of 39ºC, very close to ours, a convenient intermediary between birds and humans.

From an ecological point of view, the possibility of a same pig being infected by two different viruses, giving origin to a new rearranged lineage must be accounted. The chances are high given that, as pigs are able to be infected with the avian and human virus and live with both in farms, the introduction of human strains in pigs are often. Avian strains are also common in restricted cases of contamination by viruses such as H9N2, H3N3, H4N6, H1N1 and others.

This has already occurred in 1997 when the swine virus gained genes from an avian influenza and another from humans (our H3N2). This triple rearrangement circulates until today and it was one of the two viruses in pigs that gave origin to the Influenza A (H1N1) in 2009, directly demonstrating the potential of transmission to humans.

The 2009 pandemic has brought back attention to an important question. Pigs are transported around the world, bred in locations with a high density of animals, and – in many less developed places – are in direct contact with poultry birds and their owners. These animals have to be monitored and bred with some control, if we wish to reduce the chances of the appearance of new dangerous lineages.

Sources:

Olsen, C. (2002). The emergence of novel swine influenza viruses in North America Virus Research, 85 (2), 199-210 DOI: 10.1016/S0168-1702(02)00027-8

Landolt, G., & Olsen, C. (2007). Up to new tricks – A review of cross-species transmission of influenza A viruses Animal Health Research Reviews, 8 (01) DOI: 10.1017/S1466252307001272

Although the mutations have an important role in the diversity of the Influenza, and contribute a lot in order to develop new annual vaccines, the rearrangement causes a sudden antigenic variation (antigenic shift) that can generate a completely new virus for our immune system.

The rearrangement is a result of the Influenza cycle and of its 8 genes. When the virus enters the cell, its genes replicate in the nucleus and leave it to the cytoplasm to be encapsulated. All 8 genes have to enter the new viral particle in order for it to be infective. Therefore, each gene has a signal sequence that interacts with the M1 protein, from the structure of the viral particle. However, this signal is similar even in different viruses, and the genes from one can be encapsulated together with the genes from another.

When two different influenzas enter a same cell, its genes can be rearranged in the formation of new viral particles.

With this, in rare events (rare in relation to the infections by a single virus) in which the two different influenzas enter the same cell, different mosaics of genes can be formed in the particles that will leave. Most of these combinations are not viable, but some among the thousands that may be infectious, and an even smaller percentage can be more infectious than the original viruses. This is rearrangement.

The lottery of rearrangement, where the genes are mixed up and chosen randomly, can give origin to very dangerous Influenza lineages. Simply think about the possibility of the highly pathogenic H5N1 acquiring genes that help them spread more efficiently or, in the case of Influenza A (H1N1), acquiring genes that increase the severity of the flu it causes.

This is how the Influenza is rearranged and mixed with genes in birds, pigs and humans. And given the damage caused by these hybrid variants, this is a fundamental event in the appearance of pandemic lineages. The rearrangement has the advantage to bring new components to its genome.

Viral genes of mammals already adapted to grow in a body temperature of the host of about 37°C, against the temperature of about 40°C in birds, mix with the new avian HA and NA that will not be recognized by the antibodies. A change greater than mutations. The pigs play an important role here. Not only they live among humans and domestic birds, but also have both types of membrane receptor. Therefore, they may be infected by the avian and human virus and provide an environment with conditions where the hybrid adapts to our metabolism.

Events like this resulted in viruses that caused most of the flu pandemics. Although the H1N1 of 1918 seems to be a virus that originated directly from birds to humans (regardless having passed through pigs before it or not, it was probably transmitted as a whole virus), in 1957, a rearrangement event with an avian virus resulted in new HA and NA that allowed the virus to cause much more damage in the so-called Asian Flu. In 1968, a rearrangement occurred again where the virus acquired a new avian Hemagglutinin and caused the Hong Kong Flu.

Even rearrangement events within the same lineage are able to cause more severe diseases and failure in the coverage of the vaccine, like the flu outbreaks of 1947, 1851, 1997 and 2003. The first two were events due to H1N1 rearrangements and the last two of the human H3N2.

In 2009, we have experienced another reflex of the rearrangement, this time with the swine virus. In 1918, the avian H1N1 circulated both in humans and in pigs, generating different lineages, present until today. In 1997, a new swine virus appeared in North America, of a triple rearrangement, with a combination of human (our H3N2 generated in 1968), swine and avian Influenza genes. It is the swine H1N2. – The European pigs were virtually flu-free until 1976 when the swine H1N1 was brought in a shipment of pigs from North America. This was quickly substituted by an avian H1N1 in 1979.

Lastly, in 2008, the triple rearrangement circulating in pigs in North America rearranged again with the Eurasian H1N1 swine virus. It is not yet known if this event occurred in pigs or in humans. It is more likely that it occurred in humans, as contaminated pigs have not yet been found. This new Influenza A (H1N1) contaminated humans and is causing the current pandemic.

Sources:

Palese, P. (2004). Influenza: old and new threats Nature Medicine, 10 (12s) DOI: 10.1038/nm1141

Morens, D., Taubenberger, J., & Fauci, A. (2009). The Persistent Legacy of the 1918 Influenza Virus New England Journal of Medicine, 361 (3), 225-229 DOI: 10.1056/NEJMp0904819

The antibody connected to Hemagglutinin (HA) prevents it from linking itself to the sialic acid of the cell.

When we have the flu, in a few days the body seems to get rid of the virus. The symptoms rarely last for two weeks and, in a higher period; it normally indicates complications caused by other microorganisms. This short period of the flu is due to our immune response.

As the virus replicates in the body of infected people, the immune system captures several pieces of the viral proteins, the so-called antigens, and produces antibodies against them. There are also other types of immune response but these do not cause long-term immunity, so they are not being considered. The two largest viral targets of the antibodies are the Hemagglutinin and Neuraminidase, since they are the most exposed proteins of the virus. When the antibodies bind to them, in addition to signaling to macrophages and other types of defense cells to attack the foreign body (the virus), it can still prevent the functioning of the virus. An antibody that attacks the region of recognition of the receptor of Hemagglutinin prevents it from binding to the cells. They are called neutralizing antibodies.

Thanks to this immune response, after a few days the Influenza is unable to infect us. But how is the virus able to return?

As previously seen, when it replicates its genome, the polymerase of the Influenza causes mutations. These mutations change the composition of the viral proteins. When the aminoacids (components of the proteins) from the region where the antibody bonds (antigen) are altered in consequence of the mutation, it may lose its affinity. Therefore, as the virus infects new hosts, it accumulates small changes that in the end result in the change of its antigens. This process is called gradual antigenic drift.

Mostly, these changes do not have to be sudden to have an effect. Changes in important locations for the recognition by the antibody of only one amino acid can be enough. This was proposed by one of the most important works carried out on the immunology of influenza that changed the way in which we study the virus.

Antigenic map of hemagglutinin of H3N2 collected from 1968 (the year it infected humans) to 2003. The color of the circles defines the group to which that sample is related to. Each unit of distance between them represents the reduction of the immune response to the virus in relation to the antibodies produced against the previous one. Therefore, the greater the distance of one virus A from B, the smaller is its recognition by the antibodies against B.

Using an idea proposed in 2001, instead of only looking at the genetic difference between the samples of H3N2 causer of the human flu of different years, the authors decided to consider the immune response against them. Thus, maps are elaborated to point out how well the immune system recognizes that particular virus. The protein used in the study was the Hemagglutinin (HA) because it is against it that the most efficient antibodies are produced, reason why it is used in the annual vaccine.

Instead of a continuous change, the map showed that the virus tends to agglutinate in groups. Changes in sequences, sometimes, extensive may not represent a difference in the recognition by antibodies. On the other hand, in some cases, small changes in the HA are enough for a large antigenic distance. That is, with little mutations some Hemagglutinins can be much less recognized by the antibodies.

Genetic distance, in A and B, and antigenic, in C, isolated from H3N2. Some lineages, although genetically close, such as SI87 and BE89 in A and B, which only have one different amino acid, may not be antigenically close, as shown in C. This indicates that the antibodies produced against H3N2 in 1987 were not efficient against the 1989 virus.

It is also possible to see the change of the immune response in relation to the annual cycle. The groups remain dominant for three years in average, and the virus in this group normally appears around 2 years in advance and circulates for 2 more years after the dominance of the group.

Based on these results, the annual vaccines are seen differently. Today, the observation of the immune response to the virus of the seasonal vaccine and to the circulating virus, in addition to the genetic difference between them, is a very important factor in the production process. The research groups that monitor the Influenza and determine the lineages that will be part of the vaccine have to be carefully attentive to this fact because the change of an antigenic group of the Hemagglutinin of a virus, after it is being cultivated in eggs, can mean a failure in the immunization of a population.

Source:
Smith, D. (2004). Mapping the Antigenic and Genetic Evolution of Influenza Virus Science, 305 (5682), 371-376 DOI: 10.1126/science.1097211

Despite the negative image we have about the flu, nothing prevents it from having a certain beauty. This is how the English artist Luke Jerran transforms the way how we see the Influenza. With his glass sculptures, the virus gains an incredible and realistic beauty, once their size is smaller than the length of a light wave and they are actually translucent.

His sculpture of the Influenza A (H1N1) is very beautiful and realistic. Although it is not possible to distinguish HA from NA on the outside, we can even see the points that represent the protein M2, the viral pore. On the inside, the long filaments of the RNA of the virus are highlighted. Below, there is a bit of the production process of the HIV sculpture, which is also fantastic.

It is one of the most original forms of seeing the scientific data, fundamental phenomenon for science and for scientific dissemination. Especially in an area like virology, which deals with the interpretation of electronic microscope images (the biggest virus is not visible under an optical microscope), dealing with concepts and abstractions of difficult assimilation.

There are e-mails in circulation stating that some magazines had surprisingly predicted the epidemic of Influenza A (H1N1) years in advance. I immediately remembered an excellent book, The Coming Plague by Laurie Garrett, a journalist who won the Pulitzer Award for his work in epidemics.

In this book, besides discussing a number of very dangerous epidemics, such as Ebola and Lassa, Garrett is able to transfer the notions of public health, ecology, evolution and a number of other factors connected to infectious diseases.

In terms of flu, Laurie was the one that presented the danger of influenza, and why we know it can cause epidemics regularly, as our coexistence with animals, the mass transportation and coexistence in high concentrations can contribute to the outbreak and spread of the virus. This is why we know that new epidemics will arise.

Then, there is nothing better than her explaining this herself. It is a TED (Technology, Entertainment, and Design) lecture, an annual event to present and discuss great ideas on several areas of knowledge. This presentation is from 2007, discussing the threat of H5N1, but it applies to any severe epidemic influenza. For now, there are no legends available. On Youtube video page, it is available in a high resolution version.

Original at TED

As a free gift. Larry Brilliant discussing on what we must do to stop an epidemic, especially act quickly:

Original at TED

]]> http://blog.h1n1.influenza.bvsalud.org/en/2009/11/14/influenza-a-h1n1-the-coming-plague/feed/ 2 http://blog.h1n1.influenza.bvsalud.org/en/2009/11/06/antivirals-and-resistance-adamantanes/ http://blog.h1n1.influenza.bvsalud.org/en/2009/11/06/antivirals-and-resistance-adamantanes/#comments Fri, 06 Nov 2009 03:10:32 +0000 Atila Iamarino http://blog.h1n1.influenza.bvsalud.org/en/?p=56 In the case of a pandemic, and during the seasonal epidemics, once a person has already contracted the influenza, we can do little, other than monitor and treat him with antivirals. However, they are not always a guarantee of success, especially in the case of resistant viruses.

Amantadine and rimantadine are the first drugs used against influenza, still from a time when drugs were tested previously and before its effects were discovered [1] – it may seem strange to make this comment but the difference will soon be clarified. They act by inhibiting the action of the M2 protein.

When the Influenza enters a cell, it uses the cell’s normal metabolism for itself. Thus, to unwrap itself in the right place, its proteins are only detached from one another, releasing the genes that will invade the nucleus only when the virus is in the right place, and it measures its position through the pH of the endosome, the sack of the membrane it forms when entering.

M2 Protein serving as a channel for the H+ ions, above. Unwrapping of the virus and release of the genes, below.

When the endosome moves towards the inner part of the cell, its pH is reduced.  It is only in this acidic pH that the influenza releases its genes, ensuring that it is close to the nucleus. Protein M2 is responsible for generating this signal by forming a pore on the membrane of the virus allowing the H+ ions that generate the acidic environment to enter the virus. Amantadine makes the M2 to remain closed and prevents the formation of the pore through which the H+ ions enter, inhibiting the virus from becoming more acidic and unwrapping its proteins.

Although they are efficient, the adamantanes have some problems. The first of them is that they are neurotoxic, attacking the central nervous system as side effect. After all, our nervous system also depends on the pores that give way to ions. The second and the biggest problem is the resistance.

Amantadine in red, blocking the pore formed by M2 and preventing the H+ ions from entering the virus. With this, the M1 protein does not detach from the genes of the virus (above) and they cannot invade the cell nucleus.

Due to the type of interaction that occurs between M2 and the adamantanes, the M2 proteins can undergo mutation, which makes it resistant to these drugs without losing its activity. The most common mutation is the S31N, which means changing  a serine by an asparagine in position 31, or better still, the number 31 serine amino acid at the start of the protein, changes to an asparagine. With this change, the M2 protein loses stability when closed and it can open up and form the pore even with the amantadine attached. The virus is now resistant to these drugs and even in their presence it can unwrap itself. [2]

With the inappropriate use of the antivirals, wrong doses, prescriptions for those who do not need it, prescriptions for people with another respiratory virus, the appearance of resistant viruses is highly favored. After the appearance of the H5N1, the bird flu virus, and consequently the fear caused in the population, the search for antivirals grew considerably, giving way for this to occur more easily.

In the United States, the frequency of the adamantane-resistant influenza in the flu season at the end of 2003 – beginning of 2004 was 1.9%.  Between 2004 and 2005 it increased to 14.5% and in 2005 and beginning of 2006 it reached the alarming frequency of 92% of the circulating H3N2. Almost all due to the mutation in the residue 31. [3]

All the Influenza B are resistant to adamantanes, and in the case of influenza A (H1N1), which inherited an M2 from the swine virus circulating in Europe and Asia, amantadine and rimantadine also have no effect. [4]

Fontes:

[1]Davies, W., Grunert, R., Haff, R., McGahen, J., Neumayer, E., Paulshock, M., Watts, J., Wood, T., Hermann, E., & Hoffmann, C. (1964). Antiviral Activity of 1-Adamantanamine (Amantadine) Science, 144 (3620), 862-863 DOI: 10.1126/science.144.3620.862
[2] Pielak, R., Schnell, J., & Chou, J. (2009). Mechanism of drug inhibition and drug resistance of influenza A M2 channel Proceedings of the National Academy of Sciences, 106 (18), 7379-7384 DOI: 10.1073/pnas.0902548106
[3] Weinstock, D. (2006). Adamantane Resistance in Influenza A JAMA: The Journal of the American Medical Association DOI: 10.1001/jama.295.8.jed60009
[4] . (2009). Emergence of a Novel Swine-Origin Influenza A (H1N1) Virus in Humans New England Journal of Medicine, 360 (25), 2605-2615 DOI: 10.1056/NEJMoa0903810

From adamantine therapeutics failures, interest in developing new drugs against the flu virus had come up. Hence, appeared oseltamivir and zanamivir, neuraminidase inhibitors, the first class of planned drugs against Influenza. Here, the path taken for their production was reverse of that of amantadine. Instead of testing the drug and finding out later how it works, a possible target was identified and then the drugs were made.

Influenza virus leaving the cell. Its Hemaglutinine (HA) is still locked in the sialic acid – above. Until Neuraminidase (NA) turns the cells acid off - below.

The target was Neuraminidase (NA), an enzyme that helps liberating the virus. When the newly-formed Influenza stems from the infected cell, its Hemaglutinine (HA) connects to sialic acid from the outside, through the same mechanism used by the virus to come in. But now it must not connect to the cell but get out of it. Neuraminidase recognizes those sialic acids and cleaves them, liberating the virus.

Influenza virus leaving the cell. Its Hemaglutinine (HA) is still locked in the sialic acid – above. Until Neuraminidase (NA) turns the cells acid off - below.

The development of zanamivir and oseltamivir benefited from this step. Both drugs mimic sialic acid but are not cleaved. Thus, they are recognized by NA which connects to them and are not able to attack membrane’s acids. [1]

Oseltamivir connects to Influenza’s Neuraminidase (NA) and prevents it from cleaving the sialic acid. Thus, the virus is still locked to the cell after getting out.

Making use of this Neuraminidase affinity with sialic acid brings some advantages. First of them is the specificity, both drugs are very well recognized by viral enzyme, acting in Influenza A as well in B, and they are little recognized by human enzymes, reducing the odds of side effects. [2] Finally, as they imitate the enzyme natural substratum (molecule where it acts), loosing Neuraminidase its affinity with them implies in loosing affinity for our sialic acid. Thus, viruses with resistant NA are also less efficient, since they connect less to the drug and to the substrate. [3]

This does not mean that there are any resistant Neuraminidases. The most common resistance mutation is H274Y (or H275Y, depending on the NA type being used as reference), which means that the histidine amino acid has mutated into a tyrosine in position 275. It changes Neuraminidase’s interaction with oseltamivir, reducing its affinity to it. However, zanamivir interacts with other NA points and it is not reached by this mutation, which makes NA with H274Y resistant to oseltamivir and sensible to zanamivir.[4]

There are other resistance mutations, including some with reduced susceptibility to zanamivir [5], but generally those jeopardize much the virus’ viability. Curiously, Influenza A (H1N1) is susceptible to oseltamivir and zanamivir, while the H1N1 strain that circulated between the end of 2008 and the beginning of 2009 in the US was almost 100% resistant due to the mutation H274Y [6].

Such knowledge on resistance mutation allows foreseeing which drug should be used or not. Even before starting treatment against Influenza A (H1N1), it was already known that the most probable resistance mutation in its Neuraminidase is H274Y. Thus, it was forecast that if a virus resistant to oseltamivir appears, it would be sensible to zanamivir [7].

And that was what happened, until October 02 when 28 resistance cases were found, all with H274Y. All cases were successfully treated with zanamivir, which is not usually the first drug of choice because it is inhalable, a course damaged in flu, while oseltamivir is orally taken.

But both drugs, adamantine and sialidase inhibitors do not guarantee any comfort. In the next post of this series, I will discuss the perspectives on new antiviral development.

Sources:

[1] von Itzstein, M., Wu, W., Kok, G., Pegg, M., Dyason, J., Jin, B., Phan, T., Smythe, M., White, H., Oliver, S., Colman, P., Varghese, J., Ryan, D., Woods, J., Bethell, R., Hotham, V., Cameron, J., & Penn, C. (1993). Rational design of potent sialidase-based inhibitors of influenza virus replication Nature, 363 (6428), 418-423 DOI: 10.1038/363418a0

[2] Hata, K., Koseki, K., Yamaguchi, K., Moriya, S., Suzuki, Y., Yingsakmongkon, S., Hirai, G., Sodeoka, M., von Itzstein, M., & Miyagi, T. (2008). Limited Inhibitory Effects of Oseltamivir and Zanamivir on Human Sialidases Antimicrobial Agents and Chemotherapy, 52 (10), 3484-3491 DOI: 10.1128/AAC.00344-08

[3] De Clercq, E. (2006). Antiviral agents active against influenza A viruses Nature Reviews Drug Discovery, 5 (12), 1015-1025 DOI: 10.1038/nrd2175

[4] Collins, P., Haire, L., Lin, Y., Liu, J., Russell, R., Walker, P., Skehel, J., Martin, S., Hay, A., & Gamblin, S. (2008). Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants Nature, 453 (7199), 1258-1261 DOI: 10.1038/nature06956

[5] Hurt, A., Holien, J., Parker, M., Kelso, A., & Barr, I. (2009). Zanamivir-Resistant Influenza Viruses with a Novel Neuraminidase Mutation Journal of Virology, 83 (20), 10366-10373 DOI: 10.1128/JVI.01200-09

[6] . (2009). Emergence of a Novel Swine-Origin Influenza A (H1N1) Virus in Humans New England Journal of Medicine, 360 (25), 2605-2615 DOI: 10.1056/NEJMoa0903810

[7] Soundararajan, V., Tharakaraman, K., Raman, R., Raguram, S., Shriver, Z., Sasisekharan, V., & Sasisekharan, R. (2009). Extrapolating from sequence—the 2009 H1N1 ’swine’ influenza virus Nature Biotechnology, 27 (6), 510-513 DOI: 10.1038/nbt0609-510