Cows infected with avian influenza, how did the influenza virus become a demon? | Virus Super Topic

"There is a fish in the North Sea, named Kun. It changes into a bird, named Peng." Birds can get the flu and can infect people. Birds are the main hosts of the flu virus, but recently American cows have been infected with the avian flu virus H5N1, causing concern among local residents. Why is the flu virus so omnipresent? It has cultivated itself into a demon, with its own methods and secrets.

Written by Li Qingchao (Shandong Normal University)

Influenza viruses pose a serious threat to human health and have caused many pandemics in history. The 1918 influenza pandemic infected about 500 million people and caused 50 million deaths (some estimates put the death toll at 100 million). In 2009, influenza infected about one-fifth of the world's population, with deaths ranging from 150,000 to 570,000. It was the first "Public Health Emergency of International Concern" (PHEIC; the system was first promulgated in the International Health Regulations (2005)) recognized by the World Health Organization (WHO). It should be clear that human health may always be shrouded in the shadow of influenza pandemics.

Influenza viruses are a type of virus belonging to the Orthomyxoviridae family, and can be divided into four types: IAV (Influenza A viruses), IBV, ICV, and IDV (corresponding to A, B, C, and D in Chinese). Influenza A viruses have a wide host range, the main hosts of influenza B and C viruses are humans, and influenza D viruses have been found to infect cattle, goats, and pigs. When people talk about influenza, they generally refer to the minority of highly pathogenic influenza A viruses. Other types of influenza viruses, as well as most serotypes of influenza A viruses, are not highly pathogenic and can only cause the common cold; about 10% to 15% of common colds are caused by influenza viruses.

Does that mean that as long as we eliminate those highly pathogenic influenza viruses, we will be safe once and for all? Sorry, the "Influenza A Virus Circle" has a way of "cultivating to become a demon".

Flu viruses have different types

Influenza viruses are highly diverse and can be divided into many types.

The so-called "type" of influenza A refers to the concept of species, which is determined by the viral gene sequence. The entire influenza A virus species is also divided into different "types"; the "type" here refers to the serotype combination of influenza virus hemagglutinin (HA or H) and neuraminidase (NA or N).

Figure 1 Schematic diagram of influenza virus. Influenza virus is an enveloped RNA virus with a segmented genome. Its envelope mainly contains hemagglutinin (blue) and neuraminidase (red), and its genome is composed of 8 RNA segments (green). Image source: cdc.gov

Influenza virus is an enveloped virus. Its envelope proteins are mainly hemagglutinin and neuraminidase, which play an important role in virus infection and release. These proteins are located on the surface of virus particles and are also important targets for antibodies produced by host humoral immunity. The hemagglutinin and neuraminidase of different influenza viruses are slightly different, so the specificity of the antibodies induced is also different. If a group of pathogens have the same serotype, it means that the antibodies induced by these pathogens (mainly taken from serum for testing) have the same specificity. At present, 19 different hemagglutinin serotypes and 11 different neuraminidase serotypes of influenza A virus have been found. The combination of the two serotypes is called "type". For example, the HA of H5N1 influenza virus is 5 subtypes and NA is 1 subtype. It is not difficult to calculate that in theory, there are 18×11=198 possible types of influenza A virus. At present, more than 130 combinations of influenza A subtypes have been identified in nature, mainly from wild birds. [1]

The "type" of influenza virus has important biological significance. It reflects the host range of the virus and the severity of the disease. It is an important indicator for epidemiological monitoring and tracking of virus transmission and mutation. It also determines important issues such as vaccine design and production, and disease treatment.

Flu virus really works

The influenza virus has a wide range of hosts, and can infect everything that flies in the sky, swims in the water, and runs on the ground. Those that fly in the sky include birds and bats, those that swim in the water refer to marine mammals (not fish), and those that run on the ground include mammals including humans and ostriches (haha, so rigorous). The influenza A virus takes advantage of its host to occupy land, sea, and air.

For influenza A viruses, the most noteworthy concern is the avian influenza virus (AIV) reservoir in migratory birds, especially in wild waterfowl populations, where there are large and diverse reservoirs of influenza A viruses. Avian influenza is less harmful to birds, and most infections are usually asymptomatic. However, as birds move and migrate, avian influenza can be transmitted to animal groups such as poultry and livestock that are in close contact with humans, thereby causing human infection.

Human influenza viruses can also infect pigs. With pigs as intermediate hosts, influenza viruses from various sources infect the same pig, causing mixed infection, which may produce influenza mutants that are easily infected with humans. This virus host migration and mutation network of migratory birds → poultry → pigs → humans makes it impossible to eliminate influenza, leaving human health exposed to the threat of influenza pandemics at any time.

Figure 2 Different influenza viruses and their hosts. The main host groups of influenza A virus are wild waterfowl, poultry, pigs and humans. Different host groups have relatively fixed influenza virus types that continue to spread (dark circular arrows). Through host jumps, influenza A viruses infected by these four main host groups can infect other animals (light arrows). Image source: Reference [2]

The "weak point" of bird flu

The probability of birds contracting influenza and the diversity of viruses are both high, but it is not so easy for wild birds or poultry to directly transmit avian influenza to humans. The high-risk groups for infection with avian influenza are mainly livestock and veterinary practitioners, sales staff in live poultry markets, and slaughterers. This is because the binding and invasion of influenza viruses into cells is initiated by the specific binding between the hemagglutinin on the surface of the virus and the polysaccharides on the surface of the cell. The ability of different types of influenza viruses to infect different hosts is determined by the type and distribution of viral hemagglutinin and cell polysaccharides.

Influenza viruses use their own viral hemagglutinin (a protein that can specifically recognize polysaccharides, also known as lectin) to recognize polysaccharides on the cell surface, perform specific adsorption and initiate the invasion process. Avian influenza viruses mainly recognize and bind to sialic acid α-2,3 galactose at the end of polysaccharides (sialic acid is connected to galactose through α-2,3 chemical bonds. These two sugar molecules are located at the end of polysaccharides and can be recognized by avian influenza viruses). This polysaccharide exists in the entire respiratory and digestive tracts of birds. In contrast, sialic acid α-2,6 galactose is mainly present in the human upper respiratory tract (nasal mucosa, paranasal sinuses, pharynx, trachea) and bronchi. Sialic acid α-2,3 galactose that avian influenza viruses are obsessed with is mainly present in human bronchi and alveoli. Therefore, when an avian influenza virus "travels through mountains and rivers" and finally makes trouble in a chicken farm, it still faces the challenge of how to reach the alveoli directly when it wants to further harm humans. This can probably only happen when people and poultry are in "close contact and take a deep breath". It is not difficult to imagine that patients with avian influenza often have more serious symptoms - pneumonia (because avian influenza infects the lungs).

Once a person is infected with avian influenza by a bird, will they also transmit it to other people? This depends on whether avian influenza can be effectively transmitted between humans. In the absence of mutation, when avian influenza viruses infect new human hosts, they also face the problem of low virus attachment efficiency to upper respiratory tract tissues and difficulty in reaching cell receptors. Even if they successfully infect the upper respiratory tract, the level of viral replication of avian influenza viruses in these tissues is low. On the other hand, in order for the virus to spread effectively, in addition to being able to infect and replicate, it is also necessary to effectively spread new virus particles. When the influenza virus leaves the host cell, it will cut the polysaccharide receptors that it relies on for invasion through neuraminidase, so as to avoid the new virus being trapped on the cell surface or glycoprotein and unable to spread due to the recognition and binding of the new virus to the cell receptor. However, the neuraminidase of the avian influenza virus cannot effectively cut the human cell surface receptors, so the virus particles are poorly atomized and difficult to leave the infection site and spread through droplets. In summary, avian viruses cannot be effectively transmitted from person to person through the air.

Unlike avian influenza, the receptor of human influenza virus is sialic acid α-2,6 galactose at the end of polysaccharide. Influenza virus spread by droplets can easily find infection sites in the upper respiratory tract and establish infection. Neuraminidase can also function normally, and the new viruses produced and spread can continue to infect other cells or hosts. It is worth noting that since sialic acid α-2,6 galactose is also distributed in the bronchi and alveoli, influenza may develop into pneumonia, resulting in serious consequences.

Figure 3 Types and distribution of polysaccharides in the human respiratory tract. Sialyl α-2,6 galactose is distributed throughout the respiratory tract (shown in green), and sialic acid α-2,3 galactose is mainly distributed in the bronchi and alveoli (shown in red and dark green). Image source: Reference [3]

Figure 4 Life and death depend on a "chemical bond". A is a sialic acid α-2,6 galactose bond, and B is a sialic acid α-2,6 galactose bond. The red is sialic acid and the green is galactose. The positions of sialic acid bonded to galactose are different. Image source: Reference [4]

The "secret" of influenza virus

"Mutation" is the only way for viruses to reproduce. Point mutation and recombination are not a problem. Influenza viruses also have unique rearrangement skills. With these three mutation methods, influenza viruses may break through the original host range and achieve "host jump".

Rearrangement refers to the recombination of the genome molecules of a segmented genome virus. Influenza viruses contain a segmented genome of 8 RNA segments. If a cell is infected with two or more different influenza viruses, the RNA of both viruses will replicate in the cell nucleus. When the new virus particle is assembled on the plasma membrane, it randomly selects 8 of the two original genomes to combine, thereby producing a new viral genome. This process is called rearrangement. The new virus is called a reassortant.

The reassortment and exchange of RNA segments between mammalian influenza and avian influenza viruses causes a significant change in viral characteristics (such as antigenic "antigenic shift"), which may produce more transmissible mutant viruses that can cause influenza pandemics. In contrast, influenza viruses can also gradually adapt to new hosts through point mutations, and the antigenic changes caused by point mutations are called "antigenic drift."

Flu virus's pig teammate

With the "help" of the green snake spirit, seven gourd brothers can cultivate into one golden gourd brother. So who will help the flu virus cultivate into a demon? The answer is: pigs.

The role of bats in the process of coronavirus infection of humans is almost certain; although bats are also infected with influenza, there have been no reports of bats transmitting influenza to humans. In terms of spreading influenza, pigs can be said to be super "pig teammates" - they are important members of the transmission chain of wild birds → poultry → pigs → humans. Biologically speaking, the respiratory tract of pigs has the characteristics of polysaccharide receptors of both humans and birds: rich in two polysaccharides, α-2,6 galactose and sialic acid α-2,6 galactose. Therefore, pigs can be infected with avian influenza, human influenza and swine influenza better. When these viruses exist in pigs at the same time, there is a probability that a recombinant virus with both the transmissibility of human influenza and the pathogenicity of avian influenza will be combined. Therefore, pigs become mixers for influenza viruses, accelerating the process of viral evolution.

The 2009 H1N1 pandemic strain is a reassortment of three influenza viruses: avian, human, and swine. In this process, pigs play a key role - chickens and ducks can be infected with avian influenza from wild birds and pass it on to pigs; pigs can also be infected with human influenza viruses. When pigs are infected with two or more influenza viruses at the same time, they undergo genetic reassortment in the pig's body, producing highly virulent influenza viruses that can infect humans.

Figure 5 Important epidemic and rearrangement events in history. The 1918 influenza pandemic was caused by an avian influenza strain that originated from birds, which became prevalent in local areas again in 1977. The 2009 pandemic swine influenza virus was a virus that originated in pigs and was produced by the rearrangement of at least three influenza viruses. The main reason for the epidemic changes of seasonal influenza viruses, in addition to the changes in gene rearrangement (antigenic shift) from the natural virus reservoir of birds, is that the virus causes antigenic drift through point mutations, escaping the original human herd immunity, thus causing another epidemic. Image source: Reference [5]

How lucky does the flu virus need to be?

Currently, only the H1, H2, and H3 subtypes are naturally adapted to infect humans. For other hemagglutinins to infect humans, adaptive mutations must occur. Amino acid substitutions in the hemagglutinin receptor binding site are necessary to change the polysaccharide binding properties and promote host hopping. This process is equivalent to the process of modifying the key carried by different viruses (hemagglutinin) to open the cell lock (polysaccharide). The series of amino acid residues in the polysaccharide binding site of the viral hemagglutinin are equivalent to the teeth on the key. The specific amino acid substitutions that lead to changes in receptor binding properties are different for each hemagglutinin subtype.

To effectively infect humans, the virus must acquire a preference for human receptors, or at least have the ability to weakly bind to human receptors in addition to avian receptors, in order to successfully infect and replicate in epithelial cells in the upper human respiratory tract. To effectively spread from person to person, influenza viruses also need to reduce binding to avian receptors. This is because the human upper respiratory tract is covered with secreted mucin molecules containing alpha-2,3-linked sialic acid receptors, which cannot mediate viral invasion of cells but can bind to viral particles. Mucin molecules are tightly bound to the respiratory epithelium, making it less likely that the virus will be expelled from the respiratory tract through droplets produced by coughing or sneezing.

Viruses are a type of obligate intracellular parasites without cell structures. They only carry a small number of genes for their own use, and most of the conditions required for viral replication are provided by cells. Therefore, viruses that infect different hosts or cell types often adapt to the cell types they infect and use the conditions of the host cells to replicate. If you change a species or cell type, the virus replication process may have problems. This characteristic is called the specificity of viral infection. Viral infection specificity is a comprehensive reflection of the entire process of viral replication. The determinants that lead to host jumps of avian influenza A viruses are very complex and involve a variety of viral and host factors. The breakthrough of the aforementioned receptor is only one of the relatively important points.

The accumulation of influenza virus adaptive mutations required for avian influenza to overcome species restriction and trigger a new pandemic also includes: 1) accumulation of specific mutations in the replicase; 2) changes in affinity for receptors; 3) consistency between the virus particle fusion pH and that of the human body; 4) increased stability of virus particles; and 5) escape or resistance to restriction factors.

Figure 6: The process by which avian influenza undergoes host jump and develops into pandemic influenza or seasonal influenza. Image source: Reference [7]

How much effort do humans need to put in?

Figure 7 The spread of influenza viruses and their control. Influenza viruses can be transmitted to humans through a wide range of hosts. The source of influenza viruses is mainly wild birds, and the essential intermediate hosts are mainly poultry, pigs, and cats. The latest discovery is that cattle can also serve as "relay stations" for avian influenza.

From the evolutionary relationship between viruses and hosts, the ultimate "purpose" of viruses is not to kill the host, but to replicate themselves. New viruses that infect humans are highly pathogenic, while viruses that have a long history of coexistence with humans are often not extremely pathogenic (viruses are screened by humans, and human immune status is constantly improving. Take the new coronavirus and HIV as examples. Compared with the beginning of their appearance, the severity of the disease has decreased significantly). The avian influenza virus may eventually evolve into seasonal influenza: it continues to exist in the human population, is highly transmissible, but has poor pathogenicity. However, during this transition, a pandemic influenza may occur that causes serious disasters to humans: it is highly infectious and highly pathogenic. This is what we do not want to see.

Given the "cultivation" ability of influenza viruses, it is an important task for virologists to continue to pay attention to and curb the threat of influenza viruses to human health. Recently, it was discovered in the United States that cattle can be infected with the H5N1 influenza virus, which has aroused worldwide vigilance (cattle are usually only infected with influenza D); more seriously, it was the first confirmed case of cows transmitting the H5N1 influenza virus to humans (and cats). This reveals a major hidden danger to us: Is the avian influenza virus adapting to mammals for transmission? It has found a new way besides pigs, and may even spread among humans.

"One World, One Health". We care about the development of animal health. In addition to protecting wild animals and promoting the development of animal husbandry, we also care about the health of human beings. The spread of avian influenza in poultry and livestock will hit the breeding industry hard and cause economic losses. It is also a "window of opportunity" for human influenza pandemic. In order to control the spread of influenza, measures need to be taken at the contact surface between intermediate hosts and wild birds, intermediate hosts, and intermediate hosts and humans. For the majority of readers, getting a flu vaccine and avoiding direct contact with live poultry and wild birds are the most important preventive measures.

References

[1] https://www.cdc.gov/flu/about/viruses/types.htm

[2] Long, JS, Mistry, B., Haslam, SM et al. Host and viral determinants of influenza A virus species specificity. Nat Rev Microbiol 17, 67–81 (2019). https://doi.org/10.1038/s41579-018-0115-z

[3] Nelli, RK, Kuchipudi, SV, White, GA et al. Comparative distribution of human and avian type sialic acid influenza receptors in the pig. BMC Vet Res 6, 4 (2010). https://doi.org/10.1186/1746-6148-6-4

[4] Kuchipudi, Suresh V. et al. “Sialic Acid Receptors: The Key to Solving the Enigma of Zoonotic Virus Spillover.” Viruses 13 (2021): n. pag.

[5] Long, JS, Mistry, B., Haslam, SM et al. Host and viral determinants of influenza A virus species specificity. Nat Rev Microbiol 17, 67–81 (2019). https://doi.org/10.1038/s41579-018-0115-z.

[6] Long, JS, Mistry, B., Haslam, SM et al. Host and viral determinants of influenza A virus species specificity. Nat Rev Microbiol 17, 67–81 (2019). https://doi.org/10.1038/s41579-018-0115-z.

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