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Structure, Properties, and Subtype Nomenclature

Influenzaviruses A, B and C are very similar in overall structure. The virus particle is 80–120 nanometres in diameter and usually roughly spherical, although filamentous forms can occur. These filamentous forms are more common in influenza C, which can form cordlike structures up to 500 micrometres long on the surfaces of infected cells. However, despite these varied shapes, the viral particles of all influenza viruses are similar in composition (see figure 1 and 2). These are made of a viral envelope containing two main types of glycoproteins, wrapped around a central core. The central core contains the viral RNA genome and other viral proteins that package and protect this RNA. RNA tends to be single stranded but in special cases it is double. Unusually for a virus, its genome is not a single piece of nucleic acid; instead, it contains eight pieces of segmented negative-sense RNA, each piece of RNA containing either one or two genes. For example, the influenza A genome contains 11 genes on eight pieces of RNA, encoding for 11 proteins: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1, M2, NS1, NS2(NEP), PA, PB1, PB1-F2 and PB2 (see figure 1).

Figure 1. Structure of the influenza virion. The hemagglutinin (HA) and neuraminidase (NA) proteins are shown on the surface of the particle. The viral RNAs that make up the genome are shown as red coils inside the particle and bound to Ribonuclear Proteins (RNPs).

Figure 2. Structure of the influenza virion (A) and an Electron Microscope image of the virus (B). The hemagglutinin (HA) and neuraminidase (NA) proteins are shown on the surface of the particle. The viral RNAs that make up the genome are shown as red coils inside the particle and bound to Ribonuclear Proteins (RNPs).

Hemagglutinin (HA) and neuraminidase (NA) are the two large glycoproteins on the outside of the viral particles. HA is a lectin that mediates binding of the virus to target cells and entry of the viral genome into the target cell, while NA is involved in the release of progeny virus from infected cells, by cleaving sugars that bind the mature viral particles. Thus, these proteins are targets for antiviral drugs. Furthermore, they are antigens to which antibodies can be raised. Influenza A viruses are classified into subtypes based on antibody responses to HA and NA. These different types of HA and NA form the basis of the H and N distinctions in, for example, H5N1. There are 16 H and 9 N subtypes known, but only H 1, 2 and 3, and N 1 and 2 are commonly found in humans (see figure 3).

Figure 3. Influenza A viruses of 61 combinations of 16 HA and 9 NA subtypes out of 144 theoretical combinations have been isolated from faecal samples of ducks in Alaska, Siberia, Mongolia, Taiwan, China and Japan. So far, viruses of 83 other combinations have been generated by genetic reassortment in chicken embryos.


Pathophysiology of the Influenza Virus

The mechanisms by which influenza infection causes symptoms in humans have been studied intensively. One of the mechanisms is believed to be the inhibition of adrenocorticotropic hormone (ACTH) resulting in lowered cortisol levels. Knowing which genes are carried by a particular strain can help predict how well it will infect humans and how severe this infection will be (that is, predict the strain's pathophysiology).

For instance, part of the process that allows influenza viruses to invade cells is the cleavage of the viral hemagglutinin protein by any one of several human proteases. In mild and avirulent viruses, the structure of the hemagglutinin means that it can only be cleaved by proteases found in the throat and lungs, so these viruses cannot infect other tissues. However, in highly virulent strains, such as H5N1, the hemagglutinin can be cleaved by a wide variety of proteases, allowing the virus to spread throughout the body.

The viral hemagglutinin protein is responsible for determining both which species a strain can infect and where in the human respiratory tract a strain of influenza will bind. Strains that are easily transmitted between people have hemagglutinin proteins that bind to receptors in the upper part of the respiratory tract, such as in the nose, throat and mouth. In contrast, the highly lethal H5N1 strain binds to receptors that are mostly found deep in the lungs. This difference in the site of infection may be part of the reason why the H5N1 strain causes severe viral pneumonia in the lungs, but is not easily transmitted by people coughing and sneezing.

Common symptoms of the flu such as fever, headaches, and fatigue are the result of the huge amounts of proinflammatory cytokines and chemokines (such as interferon or tumor necrosis factor) produced from influenza-infected cells. In contrast to the rhinovirus that causes the common cold, influenza does cause tissue damage, so symptoms are not entirely due to the inflammatory response. This massive immune response might produce a life-threatening cytokine storm. This effect has been proposed to be the cause of the unusual lethality of both the H5N1 avian influenza, and the 1918 pandemic strain. However, another possibility is that these large amounts of cytokines are just a result of the massive levels of viral replication produced by these strains, and the immune response does not itself contribute to the disease.


Genetics of the Pandemic (H1N1) 2009 Virus

On April 24, 2009, the U.S. Centers for Disease Control and Prevention (CDC) determined that seven samples from suspected cases in Mexico matched the strain that had infected patients in Texas and California with no known linkages to animals or one another; the strain appeared to be spreading from human to human. The CDC determined that the strain contained genes from four different flu viruses – North American swine influenza, North American avian influenza, human influenza, and swine influenza virus typically found in Asia and Europe – "an unusually mongrelised mix of genetic sequences." A CDC investigative team arrived in Mexico City on April 25, 2009 to work with Mexican counterparts to study the virus.

Pigs are susceptible to influenza viruses that can also infect both humans and birds, so they may act as a "mixing vessel" in which reassortment can occur between flu viruses of several species. Reassortment is a process that happens if two different types of influenza virus infect a single cell and it can produce a new strain of influenza. This is because the virus genome is split between eight independent pieces of RNA, which allows pieces of RNA from different viruses to mix together and form a novel type of virus as new virus particles are being assembled. This new strain appears to be a result of the reassortment of two swine influenza viruses, one from North America and one from Europe. But the North American pig strain was itself the product of previous reassortments, and has carried an avian PB2 gene for at least ten years and a human PB1 gene since 1993. These genes were passed on to the new virus.

Gene sequences for every viral gene were made available through the Global Initiative on Sharing Avian Influenza Data (GISAID). A preliminary analysis found that the hemagglutinin (HA) gene was similar to that of swine flu viruses present in U.S. pigs since 1999, but the neuraminidase (NA) and matrix protein (M) genes resembled versions present in European swine flu isolates. While viruses with this genetic makeup had not previously been found to be circulating in humans or pigs, there is no formal national surveillance system to determine what viruses are circulating in pigs in the U.S. So far, little is known about the spread of the virus in any pig population. A preliminary analysis has also shown that several of the proteins involved in the pathophysiology of the virus are most similar to strains that cause mild symptoms in humans. This suggests that the virus is unlikely to cause severe infections similar to those caused by the 1918 pandemic flu virus or the H5N1 avian influenza.

Late on May 6, 2009, Canada's National Microbiology Laboratory first completed the sequencing of Mexican samples of the virus, publishing the result to GenBank as A/Mexico/InDRE4487/2009(H1N1). This was later shown to be nearly identical to A/California/07/2009 (H1N1), the strain from California sequenced and published by the CDC on 27 April. Samples from Mexico, Nova Scotia and Ontario had the same sequence, ruling out genetic explanations for the greater severity of the Mexican cases. The genetic divergence of the virus in samples from different cases has been analysed by Mike Worobey at the University of Arizona at Tucson, USA, who found that the virus jumped to humans in 2008 probably after June, and not later than the end of November. Worobey's research also indicated the virus had been latent in pigs for several months prior to the outbreak, suggesting a need to increase agricultural surveillance to prevent future outbreaks.

Evolutionary Potential of the Virus

On May 22, 2009, World Health Organization (WHO) Director-General Dr. Margaret Chan said that the Pandemic (H1N1) 2009 virus must be closely monitored in the southern hemisphere, as it could mix with ordinary seasonal influenza and change in unpredictable ways. Experts writing in the July issue of The New England Journal of Medicine note that historically, pandemic viruses have evolved between seasons, and the current strain may become more severe or transmissible in the coming months. They therefore stress the importance of international cooperation to engage in proper surveillance to help monitor changes in the virus's behavior, which will aid in both "vaccine targeting" and interpreting illness patterns in the fall of 2009.

Other experts are also concerned that the new virus strain could mutate over the coming months. Guan Yi, a leading virologist from the University of Hong Kong, for instance, described the new H1N1 influenza virus as "very unstable", meaning it could mix and swap genetic material (reassortment) when exposed to other viruses. During an interview he said "Both H1N1 and H5N1 are unstable so the chances of them exchanging genetic material are higher, whereas a stable (seasonal flu) virus is less likely to take on genetic material." The H5N1 virus is mostly limited to birds, but in rare cases when it infects humans it has a mortality rate of between 60% to 70%. Experts worry about the emergence of a hybrid of the more virulent Asian-lineage HPAI (highly pathogenic avian influenza) A/H5N1 strain (media labeled "bird flu") with more human-transmissible Influenza A strains such as this novel 2009 swine-origin A/H1N1 strain (media labeled "swine flu"), especially since the H5N1 strain is and has been for years endemic in birds in countries like China, Indonesia, Vietnam and Egypt.

Other studies conclude that the virus is likely well adapted to humans, has a clear biological advantage over seasonal flu strains and that reassortment is unlikely at this time due to its current ease in replication and transmission. However, Federal health officials in the U.S. noted that the horrific 1918 flu epidemic, which killed hundreds of thousands in the United States alone, was preceded by a mild "herald" wave of cases in the spring, followed by devastating waves of illness in the autumn.

As of October 2009, a research done by Taubenberger showed that the evolution of A (H1N1) is relatively slow since 1918 and the structure of the 2009 virus is similar to that of 1918 flu pandemic. A study from Hokkaido University predicted that these similar Hemagglutinin antigen residues will soon be targeted by antibody-mediated selection pressure in humans and and whether the antigenic changes similar to seasonal influenza should be the main focus of monitoring.

Antigenic Shift

Antigenic shift is the process by which at least two different strains of a virus (or different viruses), especially influenza, combine to form a new subtype having a mixture of the surface antigens of the two original strains. The term antigenic shift is more often applied specifically (but is not limited) to the influenza literature, as it is the best known example (e.g. visna virus in sheep). Antigenic shift is a specific case of reassortment or viral shift that confers a phenotypic change.

Antigenic shift is contrasted with antigenic drift, which is the natural mutation over time of known strains of influenza (or other things, in a more general sense) which may lead to a loss of immunity, or in vaccine mismatch. Antigenic drift occurs in all types of influenza including influenza virus A, influenza B and influenza C. Antigenic shift, however, occurs only in influenzavirus A because it infects more than just humans. Affected species include other mammals and birds, giving influenza A the opportunity for a major reorganization of surface antigens. Influenza B and C principally infect humans, minimizing the chance that a reassortment will change its phenotype drastically.

Antigenic shift is important for the emergence of new viral pathogens as it is a pathway that viruses may follow to enter a new niche (see figure 1). It could occur with primate viruses and may be a factor for the appearance of new viruses in the human species such as HIV. Due to the structure of its genome, HIV does not undergo reassortment, but it does recombine freely and via superinfection HIV can produce recombinant HIV strains that differ significantly from their ancestors.

Figure 1. Illustration of potential influenza antigenic shift.

Flu strains are named after their types of hemagglutinin and neuraminidase surface proteins, so they will be called, for example, H3N2 for type-3 hemagglutinin and type-2 neuraminidase. When two different strains of influenza infect the same cell simultaneously, their protein capsids and lipid envelopes are removed, exposing their RNA, which is then transcribed to mRNA. The host cell then forms new viruses that combine their antigens; for example, H3N2 and H5N1 can form H5N2 this way. Because the human immune system has difficulty recognizing the new influenza strain, it may be highly dangerous. Influenza viruses which have undergone antigenic shift have caused the Asian Flu pandemic of 1957, the Hong Kong Flu pandemic of 1968, and the Swine Flu scare of 1976. One increasingly worrying situation is the possible antigenic shift between avian influenza and human influenza. This antigenic shift could cause the formation of a highly virulent virus.

Pigs can be infected with both human and avian influenza viruses in addition to swine influenza viruses. Infected pigs get symptoms similar to humans, such as cough, fever, and runny nose. Because pigs are susceptible to avian, human and swine influenza viruses, they potentially may be infected with influenza viruses from different species (e.g., ducks and humans) at the same time. If this happens, it is possible for the genes of these viruses to mix and create a new virus (see figure 1).

For example, if a pig was infected with a human influenza virus and an avian influenza virus at the same time, an antigenic shift could occur, producing a new virus that had most of the genes from the human virus, but a hemagglutinin or neuraminidase from the avian virus. The resulting new virus would likely be able to infect humans and spread from person to person, but it would have surface proteins (hemagglutinin and/or neuraminidase) not previously seen in influenza viruses that infect humans, and therefore to which most people have little or no immune protection. If this new virus causes illness in people and can be transmitted easily from person to person, an influenza pandemic can occur.


Antigenic Drift

The immune system recognizes viruses when antigens on the surfaces of virus particles bind to immune receptors that are specific for these antigens. This is similar to a lock recognizing a key. After an infection, the body produces many more of these virus-specific receptors, which prevent re-infection by this particular strain of the virus and produce acquired immunity. Similarly, a vaccine against a virus works by teaching the immune system to recognize the antigens exhibited by this virus. However, viral genomes are constantly mutating, producing new forms of these antigens. If one of these new forms of an antigen is sufficiently different from the old antigen, it will no longer bind to the receptors and viruses with these new antigens can evade immunity to the original strain of the virus. When such a changes occurs, people who have had the illness in the past will lose their immunity to the new strain and vaccines against the original virus will also become less effective. Two processes drive the antigens to change: antigenic drift and antigenic shift, antigenic drift being the more common (see figure 1).

In the influenza virus, the two relevant antigens are the surface proteins, hemagglutinin and neuraminidase. The hemagglutinin is responsible for entry into host epithelial cells while the neuraminidase is involved in the process of new virions budding out of host cells. The host immune response to viral infection is largely determined by the immune system's recognition of these influenza antigens. Vaccine mismatch is a potentially serious problem. Antigenic Drift is the continuous process of genetic and antigenic change among flu strains.

Figure 1. Illustration of potential influenza antigenic drift.

To meet the challenge of antigenic drift, vaccines that confer broad protection against heterovariant strains are needed against seasonal, epidemic and pandemic influenza.

As in all RNA viruses, mutations in influenza occur frequently because the virus' RNA polymerase has no proofreading mechanism, providing a strong source of mutations. Mutations in the surface proteins allow the virus to elude some host immunity, and the numbers and locations of these mutations that confer the greatest amount of immune escape has been an important topic of study for over a decade.

Antigenic drift has been responsible for heavier-than-normal flu seasons in the past, like the outbreak of influenza H3N2 variant A/Fujian/411/2002 in the 2003 - 2004 flu season. All influenza viruses experience some form of antigenic drift, but it is most pronounced in the influenza A virus.

Antigenic drift should not be confused with antigenic shift, which refers to reassortment of the virus' gene segments. As well, it is different from random genetic drift, which is an important mechanism in population genetics.