INFLUENZA VIRUSES Overview
Three types of influenza viruses (A, B, and C) infect humans. Influenza virus types A and B both cause more severe symptoms than does influenza virus type C. Influenza virus A, which has several subtypes based on hemagglutinin (H) and neuraminidase (N), undergo more genetic changes than types B and C. Influenza viruses are enveloped, negative-sense segmented RNA virus that replicate in the nucleus of the infected cells. Direct droplet spread is the most common mode of transmission and the incubation is period is about 2 days. The virus multiply in ciliated respiratory epithelial cells, leading to functional and structural ciliary abnormalities, including interference with the mechanical clearance mechanism of the respiratory tract. The typical influenza illness is characterized by an abrupt onset (over several hours) of fever, diffuse muscle aches, and chills. This is followed within 12 to 36 hours by respiratory symptoms such as rhinitis, fever, myalgia, headache, cough, occasionally shaking chills, respiratory distress. The acute phase usually lasts 3 to 5 days, but a complete return to normal activities may take 2 to 6 weeks. Occasionally, patients develop a progressive viral infection causing viral pneumonia and some unusual manifestations such as CNS dysfunction, myositis, and myocarditis. The most common complication of influenza infection is bacterial superinfection usually resulting in bacterial pneumonia. Influenza virus infection can be prevented by annual vaccination, which is formulated every year because of antigenic drift that allows the virus to escape preexisting immunity from previous vaccination or infection.
INFLUENZA VIRUS GROUP CHARACTERISTICS
Influenza viruses are members of the orthomyxovirus group or family, which are enveloped, pleomorphic, single-stranded negative-sense segmented RNA viruses. They are classified into three major types, A, B, and C, based on antigenic differences in their ribonucleoprotein (NP) and matrix (M) protein antigens. Influenza A viruses are the most extensively studied because of their predominance in epidemics, and much of the following discussion is based on knowledge of this type. They generally cause more severe disease and more extensive epidemics than the other types; naturally infect a wide variety of species, including mammals and birds; and have a great tendency to undergo significant antigenic changes (Table 9–1). Influenza B viruses are more antigenically stable, are known to infect humans and seals, and usually occur in more localized outbreaks. Influenza C viruses appear to be relatively minor causes of disease, affecting humans and pigs.
✺ Influenza viruses (orthomyxoviruses) divided into three types; A, B, and C
✺ Influenza A virus has the greatest virulence and predominance in epidemic spreads because of their ability to undergo genetic changes and existence in several species
TABLE 9–1Differences Among Influenza Viruses ||Download (.pdf) TABLE 9–1 Differences Among Influenza Viruses
|FEATURE ||INFLUENZA A ||INFLUENZA B ||INFLUENZA C |
|Gene segments ||8 ||8 ||7 |
|Unique proteins ||M2 ||NB ||HEF |
|Host range ||Humans, swine, avians, equines, marine mammals, bats ||Humans, seals ||Humans, swine |
|Disease severity ||Often severe ||Occasionally severe ||Usually mild |
|Epidemic potential ||Extensive; epidemics and pandemics (antigenic drift and shift) ||Outbreaks; occasional epidemics (antigenic drift only) ||Limited outbreaks (antigenic drift only) |
Influenza A and B viruses each consist of a nucleocapsid containing eight segments of negative-sense, single-stranded RNA, which is enveloped in a lipid bilayer membrane derived from the host cell plasma membrane. The inner side of the envelope contains a layer of virus-specified matrix protein (M1). Two virus-specified glycoproteins, hemagglutinin (HA or H) and neuraminidase (NA or N), are embedded in the outer surface of the lipid bilayer envelope and appear as “spikes” over the surface of the virion. The ratio of H to N is generally 4 or 5 to 1. There is another integral membrane protein in influenza A known as M2 ion channel protein. Figure 9–1 illustrates the makeup of influenza A virus. Influenza B is somewhat similar but has a unique integral membrane protein, NB instead of M2 that is also believed to function as an ion channel. Influenza C differs from the others in that it possesses only seven RNA segments and has no neuraminidase, although it does possess other receptor-destroying capability (see further). In addition, the hemagglutinin of influenza C binds to a cell receptor different from that for types A and B.
✺ Influenza viruses are enveloped RNA virus with negative-sense segmented RNA genome
Virus-specific hemagglutinin (H) and neuraminidase (N) spikes expressed on envelope
Diagrammatic view of influenza A virus. Three types of membrane proteins are inserted in the lipid bilayer: hemagglutinin (as trimer), neuraminidase (as tetramer), and M2 ion channel protein. The eight ribonucleoproteins segments each contain viral RNA surrounded by nucleoprotein and associated with RNA transcriptase. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th edition. McGraw-Hill, 2008.)
The virus-specific glycoproteins are antigenic and have special functional importance to the virus in pathogenesis and immunity. Hemagglutinin is so named because of its ability to agglutinate red blood cells from certain species (eg, chickens and guinea pigs) in vitro. Its major biologic function is to serve as a point of attachment to N-acetylneuraminic (sialic) acid-only containing glycoprotein or glycolipid receptor sites on human respiratory cell surfaces, which is a critical first step in initiating infection of the cell.
✺ Hemagglutinin binds to receptor (sialic acid glycoprotein) on host cell for viral attachment
Neuraminidase is an antigenic hydrolytic enzyme that acts on the hemagglutinin receptors by splitting off their terminal neuraminic (sialic) acid. The result is destruction of receptor activity, which may help in preventing superinfection or aggregation of virus particle in the infected cell. Neuraminidase serves several functions. It may inactivate a free mucoprotein receptor substance in respiratory secretions that could otherwise bind to viral hemagglutinin and prevent access of the virus to the cell surface. Neuraminidase may promote fusion of the viral envelope with the host cell membrane for viral entry. More importantly, neuraminidase aids in the release of newly formed virus particles from infected cells. The newly formed virus particles aggregate on the cell surface by attaching to sialic acid through their hemagglutinins but neuraminidase removes the sialic acid from the cell surface receptor allowing the virus to be released and infect other cells. Type-specific antibodies to neuraminidase appear to inhibit the spread of virus in the infected host and to limit the amount of virus released from host cells.
✺ Neuraminidase promotes a smooth passage for the virus in the respiratory tract by inactivating mucoprotein receptors in respiratory secretions
✺ Neuraminidase has a major role in viral release from infected cells
Neuraminidase destroys viral receptor, thus preventing aggregation and superinfection in infected cells
Figure 9–2 illustrates the replication cycle of influenza virus. After viral entry in the cytoplasm of the host cells, nucleocapsids (viral RNA-protein complex) with viral RNA-dependent RNA polymerase complex (PB2, PB1, PA; see Table 9–2) move into the nucleus for transcription and replication (unique to RNA viruses). The priming of viral mRNA transcription is done by using host capped RNA primers, whereas viral RNA synthesis is performed by viral RNA-dependent RNA polymerase. Viral mRNAs are transported in the cytoplasm for protein synthesis followed by proteins translocation at various sites such as H and N on cell surface and nucleocapsid in the nucleus. Viral genomic (–) RNAs replication is carried out by viral RNA polymerase via positive-sense RNA intermediates followed by nucleocapsids assembly.
✺ Viral mRNA transcription and genomic RNA replication occur in the nucleus by using viral RNA polymerase and host cell RNA primers
Diagrammatic view of influenza virus life cycle. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th edition. McGraw-Hill, 2008.)
TABLE 9–2Virus-Coded Proteins of Influenza A ||Download (.pdf) TABLE 9–2 Virus-Coded Proteins of Influenza A
|RNA SEGMENT ||PROTEINS ||FUNCTION |
|1 ||PB2 – Polymerase component ||RNA synthesis, virulence |
|2 ||PB1 – Polymerase component ||RNA synthesis |
|3 ||PA – Polymerase component ||RNA synthesis |
|4 ||HA - Hemagglutinin ||Viral attachment |
|5 ||NP - Nucleocapsid ||RNA synthesis, binds to RNA |
|6 ||NA - Neuraminidase ||Virus release from infected cells |
|7 ||M1, M2 – Matrix protein ||Matrix, ion channel |
|8 ||NS1, NS2 – Nonstructural proteins ||NS1 is interferon antagonist |
Nucleocapsids assembly take place in the cell nucleus, but final virus assembly takes place at the plasma membrane. The ribonucleoproteins are enveloped by the plasma membrane, which by then contains hemagglutinin and neuraminidase. Virus “buds” are formed, and intact virions are released from the cell surface (Figure 9–2).
✺ Nucleocapsids assemble in the nucleus and virus assembly occurs in the cytoplasm through budding from the plasma membrane
Influenza A viruses were initially isolated in 1933 by intranasal inoculation of ferrets, which developed febrile respiratory illnesses. The viruses replicate in the amniotic sac of embryonated hen’s eggs, where their presence can be detected by the hemagglutination test. Most strains can also be readily isolated in cell culture systems, such as primary monkey kidney cells. Some cause cytopathic effects in culture.
Viral propagation and isolation in eggs and mammalian cell cultures
The most efficient method of detection is demonstration of hemadsorption by adherence of erythrocytes to infected cells expressing hemagglutinin or by agglutination of erythrocytes by virus already released into the extracellular fluid. The virus can then be identified specifically by inhibition of these properties by addition of antibody directed specifically against hemagglutinin. This method is called hemadsorption inhibition or hemagglutination inhibition (HI), depending on whether the test is conducted on infected cells or on extracellular virus, respectively. Because the hemagglutinin is antigenic, HI tests can also be used to detect antibodies in infected subjects. Research has shown that antibody directed against specific hemagglutinin is highly effective in neutralizing the infectivity of the virus.
Hemadsorption and hemagglutination inhibition can be used to detectpresence of virus
Antihemagglutinin antibodies are detectable in infected patients’ serum
✺ Hemagglutination inhibition can also be used to detect antibodies
Influenza A is considered in detail because of its great clinical and epidemiologic importance.
The influenza A virion contains eight segments of negative-sense, single-stranded RNA with defined genetic responsibilities. These functions include coding for virus-specified proteins (Figure 9–1; Table 9–2). A unique aspect of influenza A viruses is their ability to develop a wide variety of subtypes through the processes of mutation and whole-gene “swapping” between strains, called reassortment. Recombination, which occurs when new genes are assembled from sections of other genes, is thought to occur rarely, if at all. These processes result in antigenic changes called drifts (mutation) and shifts (reassortment or recombination), which are discussed shortly.
✺ Influenza A virus genome has eight negative-sense RNA segments each encoding at least one protein
✺ Mutation (antigenic drift) and reassortment (antigenic shift) produce antigenic changes in the virus
The 18 recognized subtypes of hemagglutinin (H) and 11 neuraminidase (N) subtypes known to exist among influenza A viruses that circulate in birds and mammals represent a reservoir of viral genes that can undergo reassortment, or “mixing” with human strains. Although, 16 subtypes of hemagglutinins and 9 subtypes of neuraminidases have been identified in aquatic birds, pigs are infected with two major hemagglutinins (H1 and H3) and neuraminidases (N1 and N2) and horses with two H (H3 and H7) and two N (N7 and N8). Two new subtypes, H17N10 and H18N11 have been identified in bats. Three hemagglutinins (H1, H2, and H3) and two neuraminidases (N1 and N2) appear to be of greatest importance in human infections. These subtypes are designated according to the H and N antigens on their surface (eg, H1N1, H3N2). There may also be more subtle, but sometimes important, antigenic differences (drifts) within each subtype. These differences are designated according to the major representative virus to which they are most closely related antigenically, using the place of initial isolation, number of the isolate, and year of detection. For example, two H3N2 strains that differ antigenically only slightly are A/Texas/1/77(H3N2) and A/Bangkok/1/79(H3N2).
Influenza A virus subtypes based on H and N antigens, including 18 subtypes of H and 11 subtypes of N found in various species
✺ Three subtypes of H (H1, H2, and H3) and two subtypes of N (N1 and N2) exist in humans
✺ Subtle changes known as antigenic drift (mutation) occurs in all strains, whereas drastic changes as antigenic shift (reassortment) occurs when two closely related strains of influenza A infect the same cell
Antigenic drifts within major subtypes can involve either H or N antigens, as well as the genes encoding other structural and nonstructural proteins, and may result from as little as a single mutation in the viral RNA. These mutations are caused by viral RNA polymerase enzyme because it lacks proof reading ability. The mutant may come to predominate under selective immunologic pressures in the host population (Figure 9–3). Such drifts are common among influenza A viruses, occurring at least every few years and sometimes even during the course of a single epidemic. In addition, drifts can develop in influenza B viruses but considerably less frequently.
✺ Antigenic drift occurs every year to few years with influenza A viruses, whereas antigenic shift occurs abruptly and unpredictably
Influenza virus: antigenic drift and antigenic shift. With drift, repeated mutations cause a gradual change in the antigens composing hemagglutinin, such that antibody against the original virus becomes progressively less effective. With shift, there is an abrupt, major change in the hemagglutinin antigens because the virus acquires a new genome segment, which in this case codes for hemagglutinin. Changes in neuraminidase could occur by the same mechanism. (Reproduced with permission from Nester EW: Microbiology: A Human Perspective, 6th edition. 2009.)
In contrast to the frequently occurring mutations that cause antigenic drift among influenza A strains, major changes (>50%) in the nucleotide sequences of the H or N genes can occur suddenly and unpredictably. These are referred to as antigenic shifts. (Figure 9–3 illustrates the difference between antigenic drifts and shifts). They almost certainly result from reassortment that can be readily reproduced in the laboratory. Simultaneously infecting a cell with two influenza A subtypes yields progeny that contain antigens derived from either of the original viruses. For example, a cell infected simultaneously with influenza A (H3N2) and influenza A (H1N1) may produce a mixture of influenza viruses of the subtypes H3N2, H1N1, H1N2, and H3N1. When “new” epidemic strains emerge, they most likely have circulated into animal or avian reservoirs, where they have undergone genetic reassortment (and sometimes also mutation) and then are readapted and spread to human hosts when a sufficient proportion of the population has little or no immunity to the “new” subtypes. An example was the appearance of avian influenza A (H5N1) virus in Hong Kong in 1997 that caused infection in humans. The global spread of avian influenza (H5N1 and others) continued through 1997 and onward with several more cases every year. Studies indicated that all RNA segments were derived from an avian influenza A virus, but a single insert coding for several additional amino acids in the hemagglutinin protein facilitated cleavage by human cellular enzymes. In addition, a single amino acid substitution in the PB2 polymerase protein occurred. These two mutations together made the virus more virulent for humans; fortunately, human-to-human transmission was poor as discussed further. A recent example is the emergence of swine influenza virus (H1N1) in Mexico and the southwestern United States in 2009 that contained segments from avian, human, and swine influenza A viruses, and was easily transmitted to humans and caused a severe disease, mainly in young immune-competent adults, including deaths. In 2012, a new influenza virus strain, H3N8 has been identified in the autopsies of seals, which is closely related to a strain circulating in North American birds since 2002. As this strain has the ability to target the sialic acid receptor found in the human respiratory tract, the new strain H3N8 poses risk to humans. In 2013, a new strain of avian flu (H7N9) infected humans in eastern China resulting in severe illness, including deaths. H7N9 has been found in chickens, ducks, and pigeons in live poultry markets in eastern China. H7N9 continues to cause infection and deaths in humans in China even in 2017. There is no solid evidence of human-to-human transmission because most of the infected people had contacts with sick poultry. Based on genetic analysis, H7N9 is responsive to neuraminidase inhibitors and that the virus has acquired some mutations that may allow it to infect mammals and humans.
Newly generated subtypes of influenza virus also develop mutations
H1N1 (human) and H5N1 (avian) target different regions of the respiratory tract
Additional molecular barriers limit human-to-human transmission of avian influenza virus (H5N1). One of the most important barriers is that avian and human influenza viruses target different regions of the human respiratory tract. Although the receptor for influenza viruses is sialic acid (SA) glycoprotein, there is a major difference in the sialic acid sugar positions with SA α 2,6 galactose for human influenza virus and SA α 2,3 galactose for avian influenza virus (H5N1). Human influenza virus receptor, SA α 2,6 galactose, is dominant on epithelial cells of nasal mucosa, paranasal sinuses, pharynx, trachea, and bronchi, whereas the H5N1 receptor SA α 2,3 galactose is mainly found on nonciliated bronchiolar cells at the junction between respiratory bronchioles and alveolus. It is interesting that A/Hong Kong/213/03 (H5N1) isolated from a patient recognized both SA α 2,6 galactose and SA α 2,3 galactose and is bound extensively to both bronchial and alveolar cells. More importantly, H1N1 swine influenza of 2009 was transmitted from human-to-human easily because it binds to the receptor SA α 2,6 galactose found in the upper respiratory tract, and caused greater severity because it infected the lower portion of the lungs by interacting with the receptor SA α 2,3 galactose.
Although receptors for H1N1 (human) are dominant in the upper part of the respiratory tract, H5N1 (avian) receptors are found in the lower portion of the lung in humans
H1N1 (swine) interacts with both receptors in the upper and lower respiratory tract
Major antigenic shifts, which occurred approximately every 8 to 10 years in the 20th century, often resulted in serious epidemics or pandemics among populations with little or no preexisting antibody to the new subtypes. Examples include the appearance of an H1N1 subtype in 1947, followed by an abrupt shift to an H2N2 strain in 1957, which caused the pandemic of Asian flu. A subsequent major shift in 1968 to an H3N2 subtype (the Hong Kong flu) led to another, but somewhat less severe epidemic. The Russian flu, which appeared in late 1977, was caused by an H1N1 subtype very similar to that which dominated between 1947 and 1957 (Table 9–3). The swine flu that appeared in April, 2009, in Mexico and southwestern United States was a previously unrecognized H1N1 strain, which caused a severe acute respiratory distress syndrome, including deaths, especially in young healthy immune-competent adults. Further analysis revealed that H1N1 swine influenza virus of 2009 was a reassortant that contained genetic components from four different flu viruses—North American swine influenza, North American avian influenza, human influenza, and swine influenza virus of Eurasian origin. Over the subsequent 3 months, this strain, designated swine-origin 2009 A (H1N1) rapidly spread globally. Fortunately, the pandemic tapered down in the following seasons. So, the key requirements for a pandemic influenza strain are: (1) generation of a new influenza A subtype, (2) causing a serious illness, and (3) easily transmitted from human-to-human. Although two of these three requirements were met in 2006 by H5N1, all these three prerequisites were fulfilled in 2009 by H1N1 swine. Each new human infection is an opportunity for the virus to change.
✺ Major antigenic shifts correlate with epidemics or pandemics
TABLE 9–3Major Antigenic Shifts Associated With Influenza A Pandemics, 1947-2009 ||Download (.pdf) TABLE 9–3 Major Antigenic Shifts Associated With Influenza A Pandemics, 1947-2009
|YEAR ||SUBTYPE ||PROTOTYPE STRAIN |
|1947 ||H1N1 ||A/FM1/47 |
|1957 ||H2N2 ||A/Singapore/57 |
|1968 ||H3N2 ||A/Hong Kong/68 |
|1977 ||H1N1 ||A/USSR/77 |
|1987 ||H3N2 ||No pandemic occurred; various strains of H1N1 and H3N2 continue circulating worldwide through 2008 |
|2009 ||H1N1 ||A new pandemic swine-origin H1N1 originated from Mexico followed by spread to southwestern United States |
The concepts of antigenic shift and drift in human influenza A virus infections can be approximately summarized as follows. Periodic shifts in the major antigenic components appear, usually resulting in major epidemics in populations with little or no immunologic experience with the subtype. As the population of susceptible individuals is exhausted (ie, subtype-specific immunity is acquired by increasing numbers of people), the subtype continues to circulate for a time, undergoing mutations with subtle antigenic drifts from season to season. This allows some degree of virus transmission to continue. Infectivity persists because subtype-specific immunity is not entirely protective against drifting strains; for example, an individual may have antibodies reasonably protective against influenza A/Texas/77(H3N2), yet be susceptible in succeeding years to reinfection by influenza A/Bangkok/79(H3N2). Eventually, however, the overall immunity of the population becomes sufficient to minimize the epidemic potential of the major subtype and its drifting strains. Unfortunately, the battle is never entirely won; the scene is set for the sudden and usually unpredictable appearance of an entirely new subtype that may not have circulated among humans for 20 years or more. One example we saw in 2009 was when an H1N1 swine influenza virus appeared that had not been seen previously, and the existing population had no immunity to its components.
Minor antigenic drifts allow influenza virus maintenance in population
Individual variation is significant
Humans are the major hosts of the influenza viruses, and severe respiratory disease is the primary manifestation of infection.
Why does influenza virus A possess the ability to generate new strains?
However, influenza A viruses closely related to those prevalent in humans circulate among many mammalian and avian species. As noted previously, some of these may undergo antigenic mutation or genetic recombination (reassortment) and emerge as new human epidemic strains.
✺ Human, animal, and avian strains are similar but may have differences in receptor specificities
Characteristic influenza outbreaks have been described since the early 16th century, and outbreaks of varying severity have occurred nearly every year. Severe pandemics occurred in 1743, 1889-1890, 1918-1919 (the Spanish flu), 1957-1958 (the Asian flu), 1968-1969 (Hong Kong flu), 1977-1978 (Russian flu), and 2009-2010 (Swine flu). These episodes were associated with particularly high mortality rates; the Spanish flu was thought to have caused at least 30 to 50 million deaths, and some historians estimate the worldwide toll was closer to 100 million deaths. Usually, the elderly and persons of any age group with cardiac or pulmonary disease have the highest death rate. However, the severity in 2009 swine flu was mainly seen among the young healthy adult population.
Pandemic influenza may generally have high mortality
Direct droplet spread is the most common mode of transmission. Influenza infections in temperate climates tend to occur most frequently during midwinter months. Major epidemics of influenza A usually occur at 2- to 3-year intervals, and influenza B epidemics occur irregularly, usually every 4 to 5 years. The typical epidemic develops over a period of 3 to 6 weeks, and can involve 10% of the population. Illness rates may exceed 30% among school-aged children, residents of closed institutions, and industrial groups. One major indicator of influenza virus activity is an abrupt rise in school or industrial absenteeism. In severe influenza A epidemics, the number of deaths reported in a given area of the country often exceeds the number expected for that period. This significant increase, referred to as excess mortality, is another indicator of severe, widespread illness. Influenza B rarely causes such severe epidemics. In general, human influenza viruses are not stable in the environment and are sensitive to heat, acid pH, and solvents. In contrast, avian influenza viruses (H5N1 and others) retain infectivity for several weeks outside the host. The avian virus is shed in respiratory secretions and feces, and the virus survives in the feces for a long time.
Seasonality favors winter months that may allow influenza virus to survive little longer in the environment
Epidemic intervals usually a few years
Excess mortality or increased absenteeism are indicators of epidemics
Think ➱ Apply 9-1. Because influenza A virus exists in multiple subtypes such as H1N1, H3N2, etc. in several species. Two subtypes may infect the same cell of a host such as pig or humans followed by replication and reassortment (antigenic shift) to generate more than 250 combinations. Antigenic drift may also occur in these new subtypes.
Influenza viruses have a predilection for the respiratory tract because of the presence of their receptors. They multiply in ciliated respiratory epithelial cells, leading to functional and structural ciliary abnormalities and viremia is rarely detected. This is accompanied by a switch-off of protein and nucleic acid synthesis in the affected cells, the release of lysosomal hydrolytic enzymes, and desquamation of both ciliated and mucus-producing epithelial cells. Thus, there is substantial interference with the mechanical clearance mechanism of the respiratory tract. The process of programmed cell death (apoptosis) results in the cleavage of complement components, leading to localized inflammation. Early in infection, the primary chemotactic stimulus is directed toward mononuclear leukocytes, which constitute the major cellular inflammatory component. The respiratory epithelium may not be restored to normal for 2 to 10 weeks after the initial insult.
✺ Virus multiplies in upper respiratory tract ciliated epithelial cells causing structural and functional abnormalities
✺ Block in host cell syntheses, release in lysosomal enzymes, and desquamation of ciliated and mucous producing cells
✺ Clearance mechanisms of the respiratory tract are compromised
The virus particles are also toxic to tissues. This toxicity can be demonstrated by inoculating high concentrations of inactivated virions into mice, which produces acute inflammatory changes in the absence of viral penetration or replication within cells. Other host cell functions are also severely impaired, particularly during the acute phase of infection. These functions include chemotactic, phagocytic, and intracellular killing functions of polymorphonuclear leukocytes and, perhaps, of alveolar macrophage activity.
Viral toxicity causes inflammation
Phagocytic host defenses compromised
The net result of these effects is that, on entry into the respiratory tract, the viruses cause cell damage, especially in the respiratory epithelium, which elicits an acute inflammatory response and impairs mechanical and cellular host responses. This damage renders the host highly susceptible to invasive bacterial superinfection. In vitro studies also suggest that bacterial pathogens such as staphylococci can more readily adhere to the surfaces of influenza virus-infected cells. Recovery from infection begins with interferon (α/β) production, which limits further virus replication, and with rapid generation of natural killer cells. Shortly thereafter, class I major histocompatibility complex (MHC)-restricted cytotoxic T cells appear in large numbers to participate in the lysis of virus-infected cells and, thus, in initial control of the infection. This is followed by the appearance of local and humoral antibody together with an evolving, more durable cellular immunity. Finally, there is repair of tissue damage.
Tissue damage creates susceptibility to bacterial invasion
Interferon and cytotoxic T-cell responses associated with recovery
Why is clearance mechanism of respiratory tract compromised in influenza virus infection?
Although cell-mediated immune responses are undoubtedly important in influenza virus infections, humoral immunity has been investigated more extensively. Typically, patients respond to infection within a few days by producing antibodies directed toward the group ribonucleoprotein antigen, the hemagglutinin, and the neuraminidase. Peak antibody titer levels are usually reached within 2 weeks of onset and then gradually wane over the following months to varying low levels. Antibody to the ribonucleoprotein appears to confer little or no protection against reinfection because it is an internal protein of the virus particle that cannot be recognized by circulating antibody. Antihemagglutinin antibody is considered the most protective; it has the ability to neutralize virus on reexposure because it is a surface protein of the virus easily recognized by the antibody. However, such immunity is relative, and quantitative differences in responsiveness exist among individuals. Furthermore, antigenic shifts and drifts often allow the virus to subvert the antibody response on subsequent exposures. Antibody to neuraminidase antigen is not as protective as antihemagglutinin antibody, but plays a role in limiting virus spread within the host.
✺ Antihemagglutinin antibody has protective effect
Antineuraminidase antibody may limit viral spread
Think ➱ Apply 9-2. Because the ciliated epithelial cells are damaged leading to loss of the functions of cilia.
Influenza A and B viruses tend to cause the most severe illnesses, whereas influenza C seems to occur infrequently and generally causes milder disease. The typical acute influenza syndrome is described here.
The incubation period is brief, lasting an average of 2 days. Onset is usually abrupt, with symptoms developing over a few hours. These include fever, myalgia, headache, and occasionally shaking chills. Within 6 to 12 hours, the illness reaches its maximum severity, and a dry, nonproductive cough develops. The acute findings persist, sometimes with worsening cough, for 3 to 5 days, followed by gradual improvement. By about 1 week after onset, patients feel significantly better. However, fatigue, nonspecific weakness, and cough can remain frustrating lingering problems for an additional 2 to 6 weeks.
✺ Short incubation period followed by acute disease with symptoms, including fever, myalgia, headache, and dry cough
✺ Gradual improvement within a week followed by residual and lingering problems for several weeks
Occasionally, patients develop a progressive infection that involves the tracheobronchial tree and lungs. In these situations, pneumonia, which can be lethal, is the result. Other unusual acute manifestations of influenza include central nervous system dysfunction, myositis, and myocarditis. In infants and children, a serious complication known as Reye syndrome may develop 2 to 12 days after onset of the infection. It is characterized by severe fatty infiltration of the liver and by cerebral edema. This syndrome is associated not only with influenza viruses but with a wide variety of systemic viral illnesses. The risk is greatly enhanced by exposure to salicylates, such as aspirin.
✺ Progressive viral infection may lead to pneumonia that may be lethal
✺ Reye syndrome, a serious complication, may develop 2 to 12 days after the infection
The most common and important complication of influenza virus infection is bacterial superinfection. Such infections usually involve the lung, but bacteremia with secondary seeding of distant sites can also occur. The superinfection, which can develop at any time in the acute or convalescent phase of the disease, is often heralded by an abrupt worsening of the patient’s condition after initial stabilization. The bacteria most commonly involved include Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus.
✺ Sudden worsening of symptoms suggests bacterial superinfection
✺ Bacterial superinfection includes Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus
In summation, there are essentially three ways in which influenza may cause death:
Underlying disease with decompensation. Individuals with limited cardiovascular or pulmonary reserves can be further compromised by any respiratory infection. Thus, the elderly and those of any age with underlying chronic cardiac or pulmonary disease are at particular risk.
Superinfection. Superinfection can lead to bacterial pneumonia and, occasionally, disseminated bacterial infection.
Direct rapid progression. Less commonly, progression of the viral infection can lead to overwhelming viral pneumonia with asphyxia. This phenomenon has been seen most commonly in severe pandemics; for example, the Spanish flu in 1918-1919 often produced fulminant death in healthy young soldiers and H1N1 swine flu epidemic in 2009 that caused a high fatality in young people.
Clinical manifestations of avian flu (H5N1) and swine flu (H1N1) varied with high fever, respiratory symptoms, neurologic symptoms, lymphopenia, and diarrhea. The virus replicated in the lower portion of the lung via interacting with the SA α 2,3 galactose receptor resulting in primary viral pneumonia in the absence of any secondary bacterial infection, including deaths, especially in healthy young adults. The cause of death was believed to be related to systemic dissemination, alveolar flooding, Na+ channel blockage, and cytokine storm (see Figure 7–5).
Why is H5N1, avian flu virus, not easily transmitted in humans?
During the acute phase of illness, influenza viruses can be readily detected or isolated from respiratory tract specimens, such as nasopharyngeal, nasal, and throat swabs. However, nasopharyngeal specimens typically have higher yield of virus than nasal or throat swabs. Various diagnostic tests, including virus culture, serology, rapid antigen, and molecular (viral nucleic acid) assays, immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR) are available. Rapid antigen and RNA assays take about 15 to 20 minutes, rapid immunofluorescence (antigen) assay 1 to 4 hours, real time RT-PCR1 takes 8 hours, and virus culture takes 3 to 10 days. These tests detect both influenza A and B viruses. Most strains grow in primary monkey kidney cell cultures, and they can be detected by hemadsorption or hemagglutination. Serologic diagnosis (antibody test) is of considerable help epidemiologically and is usually made by demonstrating a fourfold or greater increase in HI antibody titers in acute and convalescent specimens collected 10 to 14 days apart. For details about the HI assay, see Chapter 4.
✺ Rapid antigen detection by enzyme immunoassay and genomic RNA by PCR often used
✺ Virus can be cultured in cell lines
Antibody diagnosis is useful epidemiologically
The two basic approaches to management of influenza disease are symptomatic care and anticipation of potential complications, particularly bacterial superinfection. After the diagnosis has been made, rest, adequate fluid intake, conservative use of analgesics for myalgia and headache, and antitussives for severe cough are commonly prescribed. It must be emphasized that nonprescription drugs must be used with caution. This applies particularly to drugs containing salicylates (aspirin) given to children, because the risk of Reye syndrome must be considered.
Supportive therapy indicated
Bacterial superinfection is often suggested by a rapid worsening of clinical symptoms after patients have initially stabilized. Antibiotic prophylaxis has not been shown to enhance or diminish the likelihood of superinfection, but can increase the risk of acquisition of more resistant bacterial flora in the respiratory tract and make the superinfection more difficult to treat. Ideally, physicians should instruct patients regarding the natural history of the influenza virus infection and be prepared to respond quickly to bacterial complications, if they occur, with specific diagnosis and therapy.
Antibiotic prophylaxis does not prevent bacterial superinfection
Two classes of antiviral agents are available for use against influenza viruses—neuraminidase (N) inhibitors and viral protein M2 inhibitors (Table 9–4). Neuraminidase inhibitors include oseltamivir (Tamiflu) and zanamivir (Relenza) that were approved in 1999 and block the function of neuraminidase enzyme of both influenza A and B viruses, which is required for viral release, spread, and infectivity. The mechanism of action of these neuraminidase inhibitors is to competitively inhibit the function of the viral neuraminidase enzyme. As neuraminidase removes sialic acid from the glycoprotein receptors, the inhibitors do not cleave sialic acid residues on the surfaces of host cells and influenza viral envelopes. Therefore, viral hemagglutinin (H) binds to the uncleaved sialic acid residues, resulting in viral aggregation at the surface of the host cell and inhibition of virus release and reinfection of uninfected cells. These drugs are effective in reducing the severity of influenza virus if taken within 48 hours of onset of illness. Oseltamivir is recommended for treatment in subjects 2 weeks and older, and chemoprophylaxis in 1 year and older. Zanamivir is recommended for treatment in subjects 7 years and older, and chemoprophylaxis in 5 years and older. Zanamivir that is administered as oral inhalation is not recommended for people with underlying respiratory disease. Viral resistance has now been demonstrated for some strains of influenza A and is currently low, but this might change in the future.
✺ Neuraminidase inhibitors, oseltamivir and zanamivir, are useful for influenza A and B
Neuraminidase inhibitors competitively blocks neuraminidase activity and viral release
Resistant mutants at low frequency, but this could change in the future
Think ➱ Apply 9-3. H5N1 is not easily transmitted to humans because its receptor (SA α 2, 3 galactose) is not expressed on cells of upper respiratory tract but found in cells of lower respiratory tract.
TABLE 9–4Comparison of Antiviral Drugs for Influenza ||Download (.pdf) TABLE 9–4 Comparison of Antiviral Drugs for Influenza
|FEATURE ||AMANTADINE, RIMANTADINE ||ZANAMIVIR ||OSELTAMIVIR |
|Susceptible viruses ||Influenza A only ||Influenza A and B ||Influenza A and B |
|Emergent resistant strains ||Yes (++++) ||Yes (+) ||Yes (+) |
|Administration ||Oral ||Inhalation ||Oral |
Antivirals amantadine and rimantadine (the two symmetric amines) that were considered for influenza A treatment and prophylaxis but not for influenza B virus are not currently recommended because they have developed resistance against influenza A virus. Amantadine or rimantadine used to show a modest benefit to some patients against influenza A but not B when the drug was administered early in the illness (within 12-24 hours of onset). Unfortunately, the incidence of resistance to these amines by influenza A (H3N2), the dominant circulating strains, has risen dramatically from 0.8% before 1995 to higher than 95% by 2005. The mechanism of action of both amantadine and rimantadine was to block the ion channel of the viral M2 protein, resulting in interference with the key role for M2 protein in early virus uncoating. Later, virion assembly was also affected. Regrettably, virus resistance to both drugs can be readily developed in vitro or in vivo due to a single amino acid substitution in the transmembrane portion of the M2 protein.
Amantadine or rimantadine blocked virus uncoating and assembly
Resistance from single amino acid substitution in M2 protein
Amantadine and rimantadine not currently recommended for use due to resistance
The best available method of controlling influenza infection is to annually vaccinate all people aged 6 months and older. Although everybody older than age 6 months should be vaccinated, it is important that vaccination be directed primarily toward the elderly, individuals of all ages who are at high risk (eg, those with chronic lung or heart disease), and their close contacts, including medical personnel and household members and pregnant women.
There are two types of influenza vaccines—killed or inactivated (flu shot) and live attenuated influenza vaccine (nasal spray or FluMist). These viral vaccines are newly formulated each year to most closely match the influenza A and B antigenic subtypes [two influenza A viruses and one B virus (trivalent) or two influenza A and two influenza B viruses (quadrivalent)] currently causing infections. There are three different technologies approved by to FDA to produce influenza vaccines in the United States: (1) egg-based flu vaccine, (2) cell-based flu vaccine, and (3) recombinant flu vaccine. Egg-based flu vaccine is the most common and oldest technology used to produce both inactivated and live attenuated vaccines. The candidate vaccine viruses are grown in eggs, harvested and either inactivated (flu shot) or weakened for live attenuated vaccine (nasal spray). In the cell-based vaccine, the candidate vaccine viruses are grown in mammalian cell cultures followed by harvesting and preparing the vaccines. This method requires less time than egg-based vaccine and prevents allergic reaction with eggs to some people. The recombinant flu vaccine utilizes expression of hemagglutinin (HA), the major antigen of influenza that produces protective immune response in people, in insect cells followed by purification of the antigen. This is the fastest technology to produce influenza vaccine, free of egg allergies. Some of these vaccines also include adjuvants.
Three types of vaccines are produced in the United States; egg-based, cell-based, and recombinant
✺ Inactivated or killed (flu shot) and live attenuated vaccines (FluMist) are available
✺ Live attenuated influenza vaccine given to healthy people
There are three different types of flu shots made available recently: (1) a regular flu shot for ages 6 months and older; (2) a high-dose flu shot approved for people aged 65 years and older; and (3) an intradermal flu shot approved for people aged 18 years to 64 years. The flu shots are commonly used in two doses given 1 month apart to immunize children (aged 6 months to 8 years) who may not have been immunized previously. Among older children and adults, single annual doses are recommended just before influenza season. Vaccine efficacy is variable, and annual revaccination is necessary to ensure maximal protection. Live attenuated influenza vaccine (LAIV), which is made up of live, weakened influenza viruses (same strains used in killed vaccine) and given in the form of mist (FluMist) in the nostrils, is approved for use in healthy people 2 to 49 years of age. It is not given to pregnant women. Two weeks after vaccination, protective antibodies against influenza viruses are formed in the body that provide variable protection.
Flu vaccine recommended for ages 6 months and older and high-risk individuals
A high-dose vaccine available for ages 65 years and older
✺ Annual revaccination against most current strains is necessary to achieve protection
Two weeks after vaccination, protective antibodies are formed
A problem unique to influenza vaccinology is the inherent, often unexpected variation in antigenic drift from year to year. This often requires annual reformulation of vaccines that are hoped to provide the best protection before the onset of the next influenza season. Prediction of which strains should be used for vaccine production is based on international surveillance—always a difficult task indeed. Thus, in some years, vaccine efficacy (prevention of serologically confirmed influenza infection) has been estimated to be as high as 70% to 90%. At other times, efficacy may be only at levels estimated at 40% to 60%. After the emergence of the swine-origin 2009 A (H1N1) virus, this virus was added to annual influenza vaccine strains. The dilemma to vaccine composition will continue as new strains abruptly develop.
✺ When antigenic drift occurs unexpectedly, vaccine efficacy in the subsequent year may fall to unacceptable levels
Why does the influenza vaccine have to be reformulated every year?
A major factor contributing to this dilemma is related to difficulties in timely production of a vaccine. Up until very recently, all available vaccines had to be prepared in embryonated hen’s eggs—a cumbersome process that required at least 22 weeks of preparation. There are new methods whereby new strains can be identified quickly and mass produced in mammalian cell culture (as described earlier) instead of eggs, thus reducing the production time by as much as 50% with far higher vaccine quantities. In addition, the cell-based vaccine could be given to people with egg allergies.
Switching vaccine production from hen’s eggs to cell cultures would greatly improve responses to both epidemic and pandemic threats