Lecture 20 INFLUENZA (Influenza virus A)
Reading Assignments: (1) Text Chapter 36; (2) Laver, W. G., N. Bischofberger, and R.G. Webster. Disarming Flu Viruses. Scientific American (January 1999), (3) The Role of Neuraminidase Inhibitors in Influenza – Second International Symposium on Influenza and Other Respiratory Virsues, December 12, 1999. (4) Gilligan, P.H., M.L. Smiley, and D.S. Shapiro. 1997. Cases in Medical Microbiology and Infectious Diseases (2nd ed.), (Case #11) pp. 53-56. American Society for Microbiology, Washington, D.C.
1. CLASSIFICATION- "myxovirus"- an affinity for mucins (see Table 39-2)
a. Orthomyxoviridae - Influenza virus types A B C (the orthomyxoviruses)
b. Paramyxoviridae- Parainfluenza viruses 1-4, respiratory syncytial virus, mumps virus, measles virus (the paramyxoviruses)
2. STRUCTURE OF INFLUENZA VIRUSES (See Table 39-1 Important properties of orthomyxoviruses)
A. Virion- spherical, pleomorphic, 80-120nm in diameter (helical nucleocapsid, 9 mm in diameter)
B. Composition: (RNA 1%, protein (73%), lipid (20%), carbohydrate (derived from the host, 6%)
C. Genome: Single-stranded RNA, segmented (8 molecules, negative-sense)
D. Viral Proteins- includes structural (form part of the virion) and nonstructural (the enzyme needed for replication) proteins.
1. Nucleoprotein (NP) (a basic nucleic acid binding protein)- associates with viral RNA to form a helical nucleocapsid (RNP).
2. Three large proteins (PB1, PB2, and PA) are bound to the viral RNP. Responsible for RNA transcription and replication (RNA dep. RNA polymerase complex).
3. Matix proteins:
a. (M1) protein- an amorphous layer which links the ribonucleoprotein core to the overlying lipid envelope. (The most abundant particle in the virus-40% of total viral protein)
b. The M2 matrix protein - a membrane channel which allows entry of hydrogen ions into the nucleocapsid, facilitating uncoating.
E. Envelope:
1. A lipid envelope derived from the host cell membrane; sugars on the glycosylated proteins are also from the host.
2. Embedded in the envelope - two virus-encoded glycoprotein spikes: The hemagglutinin (25% of viral proteins) and neuraminidase (5% of viral proteins) proteins are the most important determinants of virulence. Changes in these two surface glycoprotein antigens determines antigenic variation in influenza viruses.
a. Hemagglutinin (HA)
i. Structure and Nomenclature
a. HA molecule consists of two subunitsHA1 and HA2) folded into a complex structure forming an elongated stalk capped by a large globule. The base of the stalk anchors it into the membrane.
b. Three HA monomers associate to form a HA trimer
c. Five antigenic sites on each HA monomer undergo change; variability in these regions is responsible for the continual evolution of new strains and influenza epidemics. Other regions of the HA molecule are conserved.
d. The HA molecule is the major antigen against which neutralizing antibody is directed.
e. Derives its name because it agglutinates certain types of red blood cells (chicken).
ii. Functions
a. Viral Attachment Protein –(the 1st step in the viral replication cycle)- binding of the virion to the host cell glycoprotein receptor - sialic acid (N-acetyl neuraminic acid) on epithelial cells of the respiratory tract.
b. Promotes fusion of the envelope to the cell membrane
b. Neuraminidase (NA)
i. Structure
a. Tetramer spike composed of four identical monomers. A
slender stalk topped with a box-shaped head. The
catalytic site for neuraminidase is on the top of each
head (each NA spike contains four acitve sites.)
b. Neuraminidase is also antigenic, and antibodies to NA
decrease the efficiency of viral spread.
ii. Functions
a. In the last step of the replication process as a hydrolytic enzyme - removes sialic acid and facilitates the release of the virus from the infected cell surfaces during the budding process.
b. ? May also function in the beginning of the replication cycle by cleaving sialic acid resides of mucus - allowing the virions to reach the target tissues, the respiratory epithelial cells.
3. REPLICATION OF INFLUENZA VIRUSES (See Fig. 39.4) -know the steps in influenza virus replication)
A. Viral attachment to sialic acid-containing receptors on epithelial cells via HA.
B. Entry into the cell by endocytosis.
C. Fusion of the viral envelope with the endosomal membrane and release of the RNPs (nucleocapsids) into the cytoplasm (uncoating) :
1. Acidification of the endosome causes a conformational change in HA to expose hydrophobic fusion-promoting regions of the protein. The viral envelope fuses with the endosomal membrane, triggering uncoating.
2. It is believed that the M2 ion channel protein permits entry of ions from the endosome into the virus particle, triggering the conformational change in HA.
3. Understand how the anti-viral drug amantadine interferes with this uncoating process.
D. Viral nucleocapsids travel to the nucleus (along with RNA dep. RNA polymerase).
E. RNA dep RNA polymerase transcribes the 8 segments of negative sense RNA into 10 positive sense mRNAs. (Unusual for an RNA virus to be transcribed in the nucleus. Why? Influenza virus scavenges the capped 5 termini of cellular RNA' s (the sequence needed for binding to the ribosomes) for its mRNAs.
F. mRNA's travel to the cytoplasm and are translated into structural and nonstructural proteins. The HA and NA proteins are processed by the endoplasmic reticulum and Golgi apparatus and are glycosylated during their journey to the cell surface where they are inserted into the cytoplasmic membrane. M proteins associates with the inner surface of the cytoplasmic membrane where HA and NA are located.
G. Meanwhile, the NP and P proteins (RNA dep. RNA pol.) are transported back to the nucleus for replication of the genome. The RNA dep RNA polymerase synthesizes 8 full length positive templates from each of the 8 negative sense genomic RNAs. The positive templates are used to synthesize 8 full length negative sense progeny genomes. The progeny genomes associate with NP and P proteins to form nucleocapsids which travel back to the cytoplasm.
H. Nucleocapsids travel to the inner surface of the cell and associate with the matrix proteins and regions of the cytoplasmic membrane containing HA and NA proteins.
I. The progeny virions bud from the plasma membrane, 8 hours after the virion entered the cell. (Numerous defective particles and a few complete virons are formed.)
4. CLASSIFICATION AND NOMENCLATURE OF INFLUENZA VIRUSES
A. Influenza Types
Antigenic differences in 2 internal structural proteins (the NP, and M proteins) are used to divide influenza viruses into 3 types- A, B, and C:
1. Influenza A- highly variable antigenically ; responsible for most cases of epidemic influenza
2. Influenza B- exhibits antigenic changes; sometimes causes epidemics
3. Influenza C- antigenically stable; causes only mild illness
B. Complete Nomenclature System for Influenza A viruses
Influenza A viruses are further classified into subtypes based on the antigenicity of their HA and NA molecules. A uniform classification scheme has been adopted internationally to describe each new isolate:
1. Animal isolates: Type, host of origin, geographic origin, strain number, and year of isolation, HA and NA subtype. Ex: A/swine/Iowa/15/30(H1N2)
2. Human Isolates: Type, geographic origin, strain number, year of isolation, HA and NA subtype. Ex: A/Hong Kong/03/68(H3N2). 14 subtypes of HA (H1-14) and nine subtypes of NA (N1-N9), in many different combinations have been recovered from humans, swine, horses, and birds. The subtypes seen in human infections are H1, H2, and H3, and N1 and N2.
5. ANTIGENIC VARIATION IN INFLUENZA A VIRUSES: Antigenic Drift, Antigenic Shift, and most recently, “Jumping The Species Barrier”. A unique aspect of Influenza A viruses are their ability to develop a wide range of subtypes through mutation and reassortment of the two envelope proteins, HA and NA. Antigenic variants of influenza virus have a selective advantage over the parental virus in the presence of antibody directed against the original strain. (No other respiratory tract virus exhibits significant antigenic variation like Influenza A virus!)
A. Antigenic Drift-a slight change in either the HA or NA protein; due to the accumulation of point mutations in the gene which results in amino acid changes in the protein. These sequence changes can alter antigenic sites on the molecule and allow the virion to escape recognition by the host's immune system. Sufficient antigenic changes may occur to cause epidemics of influenza A.
B. Antigenic Shift- major changes in the HA and NA proteins (changes too extreme to be explained by mutation). Mechanism is genetic reassortment in which the segmented genomes of influenza viruses reassort readily in cells that are coinfected with human and nonhuman influenza viruses, especially those of avian origin. Antigenic changes are so extensive the whole population is susceptible and a pandemic may result.
1. Evidence- Human and animal strains reassort to generate hybrid progeny in the laboratory. (Ex. A (H3N2) + A (H1N1)------Progeny H3N2, H1N1, H1N2, H3N1.
2. Major antigenic shifts occurs every 8-10 years.
There have been at least 4 pandemics in the past century- each characterized by a new combination of HA and NA:
The 1918 “Spanish flu” was H1N1 (See Reading Assignment 3)
The 1957 “Asian flu” was H2N2
The 1968 “Hong Kong flu” was H3N2
The 1977 “Swine flu” was H1N1
3. Recent Epidemiology: Since 1977, there have been two major subtypes of
Influenza A circulating in the world (Influenza B has continuously circulated) :
a. “Hong Kong flu” (H3N2) - present since emerged in a pandemic form in 1968.
b. “ Swine flu” (H1N1) emerged in 1977 – new pandemic strain unable to totally replace the circulating A (H3N2) subtype. Both continue to circulate.
4. Evidence That Pigs are the “Mixing Vessels” - allowing influenza A to be transmitted from birds ----- pigs ----man. The last 3 major shifts in influenza A originated in China, where much of the population is rural and in close contact with ducks and pigs. Influenza viruses multiply in the cells lining the intestinal tract of ducks and are shed in high concentrations into water, where they remain viable for days or weeks. Pigs can acquire the infection by drinking contaminated water. Recent experiments published in J. Virol. 72: 7367- 7373 (1998) demonstrate that the epithelial cells that line the throats of pigs contain receptors for both human and avian influenza viruses. Thus, mixed infections with avian and human viruses (with reassortment of HA and NA) can occur in the same cell, creating new viruses that reinfect the human population.
C. Antigenic Variation by “Jumping the Species Barrier”- The threat of H5N1 and
H9N2 Influenza A viruses.
1. In 1997, direct transmission of avian influenza viruses from birds to humans occurred in Hong Kong. Influenza A virus subtype H5N1 jumped from chickens to man causing 18 cases and 6 deaths. At the time, it was believed that no person-to person spread occurred. Recent retrospective serology now indicates that health care workers and close contacts of patients were probably infected- but transmission was inefficient. The virus would have to adapt further to the human host to spread easily from human to human.
2. In 1999, and avian H9N2 virus was transmitted to two children in Hong Kong. Both had weak physiques, had mild disease and recovered.
D. When Will the Next Influenza Pandemic Occur?
“The influenza clock is ticking, but we don't know what time it is”.
6. EPIDEMIOLOGY OF Influenza A virus Infections in Humans
A. Reservoir
While humans are the major reservoir, influenza A strains closely related to humans circulate among many avian and mammalian species. Sometimes the animals carry the virus without symptoms. In other cases, the animals have influenza-like disease.
B. Transmission
Person to person -via aerosols -direct droplet spread by inhalation; young children are the most efficient transmitters of infection.
C. Seasonality in temperate climates – December to March
7. CLINICAL FINDINGS (SYMPTOMS OF DISEASE)
A. Acute Uncomplicated Influenza A (in adults)- Influenza A is a localized infection of the upper and lower respiratory tract. After a short incubation period of 1-2 days, symptoms appear abruptly and include chills, headache, and dry cough, followed by high fever, muscular aches, malaise and anorexia. Fever, respiratory and systemic symptoms typically last 3-4 days. Cough and weakness may persist for 1-3 weeks.
B. Acute Influenza A (in chidren) -Symptoms in children may be more severe and include higher fevers and gastrointestinal symptoms such as vomiting. Febrile convulsions can occur. Myositis, otitis media, sinusitis, and croup may also be seen. (Croup - an infection in the upper respiratory tract that may cause swelling in the larynx and a persistant barking cough.)
C. Complications of Influenza A infections:
a. Pneumonia- Serious complications occur in the elderly and debilitated, especially those with underlying cardiopulmonary or other chronic disease. Pneumonia may be viral (interstitial pneumonia), secondary bacterial, or both. Increased mucous secretions help carry viruses and bacteria into the lower respiratory tract. Loss of ciliary clearance, dysfunction of phagocytic cells, and provision of a rich bacterial growth medium by alveolar exudate may lead to bacterial superinfection. Bacterial pathogens most often involved are S. aureus, S. pneumoniae, and H. influenzae.
b. Reye's Syndrome- acute encephalitis of children and adolescents. Hepatic dysfunction seen – 40% mortality. Possible relationship between aspirin and this complication of influenza.
8. PATHOGENESIS
A. Effect on Individual Host Cells:
Influenza A is a lytic virus; multiplication in cells leads to lysis and cell death.
B. Influenza – An Example of a Localized Viral Infection of the Mucosal Membrane
(Review Lectures 5 and 6) with cellular destruction and desquamation of the superficial mucosa of the respiratory tract. The principal site of replication is the columnar epithelial cells, but viral replication can occur throughout the entire respiratory tract. Infected ciliated columnar cells become vacuolated and lose their cilia, and mucosal and ciliated eipthelial cells become necrotic and desquamate. The basal layer of epithelial tissue is not affected, and viruses are rarely recovered from the blood. (Damage to the respiratory epithelium destroys the host's nonspecific defenses allowing for secondary bacterial infections.) An inflammatory reaction occurs- primarily macrophages and lymphyocytes (not neutrophils). Interferon is induced within 1 day of viral shedding (Review Lectures 5 and 6 and 8 for host defenses against viral infections. Know how interferon works!). In uncomplicated infections the fever and muscle aches are much greater than the clinical symptoms and pathological changes.) Why? It is believed that the release of IL-1 (interleukin 1) from macrophages results in fever, while interferon (which is quickly induced) is responsible for the muscular aches and fatigue. A variety of inflammatory mediators produce vasodilation and edema throughout the respiratory tract. Regeneration of the respiratory epithelial cells takes 3-4 weeks.
9. HOST RESPONSES: NONSPECIFIC AND SPECIFIC IMMUNITY
Recovery from influenza infections is associated with:
A. Nonspecific Immunity – the production of interferon (Review Lecture 5 and 6 and 8)
B. Specific Immunity
1. A Mounting Cell Mediated Immune Response
Recent animal studies have shown that both CD4+ and CD8+ T cells contribute to immunity. Animals that are deficient in both CD4+ and CD8+ cells do not survive influenza, but those deficient in only CD4+ or CD8+ are able to clear the virus.
2. The Humoral Immune Response
a. Antibodies to HA are important in:
(1) neutralization of virus- prevention of viral attachment
(2) complement activation (via the classical pathway)
(3) ADCC (see lecture 8)- NK cells kill virus infected cells with the help of antibody).
Resistance to reinfection with influenza viruses is correlated with serum and secretory IgA molecules. Antibody modifies the course of the disease; a person with low titers of antibody may become infected but they will experience a mild form of the disease.
b. Antibodies to NA are inefficient in neutralizing influenza viruses, but they restrict virus release from infected cells, reduce the intensity of infection and enhance recovery.
10. LABORATORY DIAGNOSIS
A. Isolation and identification of virus
1. Specimens- nasal washings and throat swabs during acute phase
2. Isolation method- embryonated eggs and monkey kidney cells. Screen for the presence of influenza virus by hemagglutination (adding RBC's to culture fluid – get clumping. See Prescott Fig. 32.1, p. 666)
3. Identification and subtype determination by hemagglutination inhibition (See Prescott Fig. 32.1, p.666) using antisera against currently prevalent strains.
B. Serology of acute and convalescent patient sera using hemagglutination inhibition and complement fixation. A four fold increase in titer must occur to indicate influenza infection.
11. Treatment and Prophylaxis WITH Antiviral Drugs
A. Amantadine hydrochloride (or rimantadine) are used to prevent and treat Influenza A infections. These drugs block uncoating of virus in the host cell and prevent viral replication.
Mechanism: After receptor-mediated endocytosis, the acid pH of the endosome causes a conformational change in the HA to allow fusion of the viral envelope with the membrane of the endosome. The M2 ion channel protein present in the virion permits the entry of the ions from the endosome into the virus particle, triggering the conformational change in HA. The primary antiviral action of amantadine is to block the acid-activated channel by the viral M2 protein – effectively inhibiting the uncoating of the virus. (Resistance to amantadine is associated with a single amino acid substitution in the RNA which codes for the transmembranous region of the M2 protein.)
Amantadine (and rimantadine) are most effective when given prophylactically, before an individual is exposed to the virus, leading to a 70-80% reduction in symptoms. Drug treatment should be used for high risk individuals if they have not been vaccinated or if a new influenza A strain is epidemic.
B. Neuraminidase Inhibitors (See Reading Assignments 2 and 3)
1. Zanamivir – delivered by inhalation
2. Oseltamivir – delivered orally (Referred to as GS4104) in Reading Assignment (2)
12. Prevention and Control by Vaccination
Inactivated vaccines are used to prevent influenza in the U.S, however the vaccines are continually being rendered obsolete as viruses undergo antigenic drift and shift. Vaccines are whole virus (WV), or subvirions (SV) (purified virus disrupted with detergents) or surface antigens (composed primarily of HA and NA antigens. Vaccines are 75-80% effective but yearly vaccination is required to afford maximal protection. Vaccination is contraindicated in persons with allergies to egg protein. The vaccine is usually a cocktail containing 2 type A viruses and a type B virus of the strains isolated the previous winter. Federal agencies and the WHO recommend each year which strains should be included in the vaccine. Recent phase III clinical trials of an intranasal influenza vaccine did not result in FDA approval of the vaccine.