MEDICAL MICROBIOLOGY, PAMB 650/720
Dr. Margaret Hunt
FALL 2008 LECTURES: 66-67
REFERENCE: Murray et al., Microbiology, 5th Ed., Chapter 60 WEB SITES:
http://www.cdc.gov/flu/ http://www.cdc.gov/mmwr/PDF/rr/rr5707.pdf - ACIP recommendations for 2008 http://www.cdc.gov/flu/professionals/vaccination/#summary - for links to latest details on vaccination http://www.ninds.nih.gov/disorders/ - resource for neurological diseases including Guillain Barre syndrome - from the National Institute for Neurological Disorders and Stroke (NINDS) http://www.ninds.nih.gov/disorders/gbs/gbs.htm - Guillain-BarrĂŠ syndrome information from the NINDS site http://www.ninds.nih.gov/disorders/reyes_syndrome/reyes_syndrome.htm Reye's syndrome NOTE: The spaces in the above links are underline characters. TEACHING OBJECTIVES: Brief review of influenza virus structure and properties. Discussion of influenza virus pathogenesis and disease, genetics, epidemiology, prevention and treatment. Discussion of the role of interferons in viral infection.
GENERAL True influenza is an acute infectious disease caused by a member of the orthomyxovirus family (influenza virus A, B or to a much lesser extent influenza virus C). However, note that the term 'flu' is often loosely used for any febrile respiratory illness with systemic symptoms that may be caused by many different bacterial or viral agents. Influenza outbreaks usually occur in the winter in temperate climates.
South Carolina 1996-1997 DHEC bulletin malathia influenzae per le stelle
influenza A influenza B Influenza virus CDC website
ORTHOMXYOVIRUSES HA - hemagglutinin NA - neuraminidase helical nucleocapsid (RNA plus NP protein) lipid bilayer membrane polymerase complex
type A, B, C : NP, M1 protein sub-types: HA or NA protein Disease potential: Major outbreaks are associated with influenza virus type A or B. Infection with type B influenza is usually milder than type A. Type C virus is associated with minor symptoms. Proteins: The internal antigens (M1 and NP proteins) are the type specific antigens used to determine if a particular virus is type A, B or C. The M1 proteins of all members of each type show cross reactivity. The NP proteins of all members of each type show cross reactivity. The external antigens (HA and NA) show more variation and are the subtype and strain specific antigens. These are used to determine the particular strain of e.g. influenza A responsible for an outbreak. PATHOGENESIS AND DISEASE: Spread: Person to person spread is primarily via small particle (less than 10Âľm diameter) aerosols that can get into the respiratory tract. The virus can also survive for a short time on surfaces and can be spread by this route if virus is introduced to the nasal mucosa before the virus loses infectivity. The incubation period is short: 18-72 hours. The virus concentration in nasal and tracheal secretions remains high for 24-48 hrs after symptoms start, may last longer in children. Titers are usually high, so there are enough infectious virions in a small droplet to start a new infection. Site of infection: Influenza virus infects the epithelial cells of the respiratory tract. The cells die â€“ in part due to the direct effects of the virus on the cell, also possibly due to the effects of interferon, and also due to cytotoxic T-cells at later times. The efficiency of ciliary clearance is thus reduced, this leads to impaired function of the mucus elevator, and thus there is reduced clearance of infectious agents from the respiratory tract. Gaps in the protective epithelium provide other pathogens with access to other cells. Viremia is very rare. Recovery: Interferon may play a role by decreasing virus production. Many of the symptoms of uncomplicated influenza (muscle aches, fatigue, fever) are associated with the efficient induction of interferon. The cell-mediated immune response is important in viral clearance. The antibody response is usually not significant until after virus has been cleared. Repair of the respiratory epithelium begins rapidly, but may take some time to complete.
(time course will vary from virus to virus) Interferons play an important role in the first line of defense against viral infections. They are part of the non-specific immune system and are induced at an early stage in viral infection – before the specific immune system has had time to respond. Interferons are made by cells in response to an appropriate stimulus, and are released into the surrounding medium; they then bind to receptors on target cells and induce transcription of numerous genes in the target cells. This results in an anti-viral state in the target cells. TYPES OF INTERFERON: TYPE I interferon: Interferon-α (leukocyte interferon) is produced by virus-infected leukocytes, etc Interferon-β (fibroblast interferon) is produced by virus-infected fibroblasts, or virusinfected epithelial cells, etc Interferon-α (a family of about 20 related proteins) and interferon-β are particularly potent as antiviral agents. They are not expressed in normal cells, but viral infection of a cell causes interferons to be made and released from the cell (that cell will often eventually die as a result of the infection). The interferon binds to target cells and induces an antiviral state. Both DNA and RNA viruses induce interferon but RNA viruses tend to induce higher levels. Double-stranded RNA produced during viral infection may be an important inducing agent. Other stimuli also cause these interferons to be made: e.g. exogenous double-stranded RNA, lipopolysaccharide, other components of certain bacteria. TYPE II interferon Interferon-γ (immune interferon) is produced by certain activated T-cells and NK cells. Interferon-γ is made in response to antigen (including viral antigens) or mitogen stimulation of lymphocytes. INTERFERON-α AND INTERFERON-β (TYPE I INTERFERONS) These interferons induce numerous proteins, and the function of many of these is not fully understood. However, three of the proteins that appear to play an important role in the induction of the anti-viral state have been intensively studied. Expression of one of these proteins (2’5’ oligo A synthase) results in activation of the second of these proteins (a ribonuclease) which can break down mRNA, and expression of the third protein (a protein kinase) results in inhibition of the initiation step of protein synthesis. These activities target viral protein synthesis, but also result in inhibition of host protein synthesis. Thus it is important that these proteins are only made and activated when needed.
Interferon treatment induces synthesis of the inactive form of these proteins in the target cell. Double-stranded RNA is needed for activation of these proteins. It directly activates 2’5’ oligo A synthase and protein kinase R, and indirectly activates ribonuclease L (since this needs 2'5'oligo A, the product of 2’5’ oligo A synthase, for activation). Thus these potentially toxic pathways are only activated in the interferon-treated cell if double-stranded RNA is made, this will usually only happen if virus infection actually occurs. The activation of these proteins may sometimes result in the death of the cell, but at least the progress of the infection is prevented. interferon-alpha, interferon-beta interferon receptor
induction of 2’5’oligo A synthase ds RNA
induction of ribonuclease L
activated 2’5’oligo A synthase
induction of protein kinase R (PKR)
activated ribonuclease L
activated protein kinase R ATP
Phosphorylated initiation factor (eIF-2)
inhibition of protein synthesis
OTHER EFFECTS OF INTERFERONS The pathway described above is by no means the only way that interferons protect cells against viruses and other pathogens. All three interferons increase expression of class I MHC molecules and thus promote recognition by cytotoxic T cells. All three interferons can activate NK cells which can then kill virus-infected cells. Interferon-γ increases expression of class II MHC molecules on antigen-presenting cells and thus promotes presentation of antigens to helper T cells. Interferon-γ can also activate the ability of macrophages to resist viral infection (intrinsic antiviral activity) and to kill other cells if they are infected (extrinsic antiviral activity). Interferons have many other effects on gene expression, not all of which are understood. THERAPEUTIC USES OF INTERFERONS One currently approved use for various types of interferon-α is in the treatment of certain cases of acute and chronic hepatitis C and chronic hepatitis B. Interferon-β is approved for use for certain types of multiple sclerosis. Interferon-γ has been used to treat a variety of diseases in which macrophage activation might play an important role in recovery, eg. lepromatous leprosy, leishmaniasis, toxoplasmosis.
Since interferons have anti-proliferative effects, they have also been used to treat certain tumors such as melanoma and Kaposiâ€™s sarcoma. SIDE EFFECTS OF INTERFERONS Common side effects of interferons: fever, malaise, fatigue, muscle pains High levels of interferons can cause kidney, liver, bone marrow and heart toxicity. VIRAL DEFENSES AGAINST THE NON-SPECIFIC AND SPECIFIC IMMUNE SYSTEMS Not surprisingly, some viruses have developed defenses against the interferon-induced antiviral response and other aspects of the immune defense system. For example, viruses may code for proteins which block interferon binding to cells, inhibit the action of the interferon-induced protein kinase, inhibit NK function, interfere with cell surface expression of MHC, block complement activation, prevent the host cell committing apoptosis, etc.
Protection: Humoral response: IgG and IgA are important in protection against re-infection. IgG persists longer than IgA, so plays a more important role in long term immunity. Antibody to the HA protein is most important since this can neutralize the virus and prevent infection. (Neutralization: the antibody binds an exposed component of the virion and prevents the virus initiating an infection. Neutralization frequently involves blocking of binding to host cell receptors but may work at other steps involved in the entry and uncoating of viruses.) Antibody to the NA protein has some protective effect since it seems to slow the spread of virus. Clinical findings: Usually most severe in the very young (under 5 yrs) and the elderly. Young children often lack of antibodies to influenza (no prior exposure); also small diameter of respiratory tract components means inflammation and swelling can lead to blockage of parts of respiratory tract, sinus system and Eustachian tubes. Although children with risk factors for influenza complications have a higher case fatality rate, the majority of pediatric deaths occur among children with no known high-risk conditions. The elderly population often have underlying decreased effectiveness of the immune system and/or chronic obstructive pulmonary disease or chronic cardiac disease.
1. Uncomplicated Fever (38-40°C) Myalgias, headache. Ocular symptoms - photophobia, tears, retro-orbital ache. Dry cough, nasal discharge. 2. Pulmonary complications, sequelae: Croup (acute laryngotracheobronchitis) in young children – symptoms include cough (like a barking seal), difficulty breathing, stridor (crowing sound on inspiration) Primary influenza virus pneumonia Secondary bacterial infection, typically: Streptococcus pneumoniae Staphylococcus aureus Hemophilus influenzae The build up of fluids and lack of mucociliary clearance in the respiratory tract provide a good environment for bacterial growth. Complications often occur in patients with underlying chronic obstructive pulmonary disease or heart disease. The underlying problems may not have been recognized prior to the influenza infection. 3. Non-pulmonary complications: myositis (rare, more likely to be seen in children after type B infection) cardiac complications encephalopathy – increased US surveillance of patients younger than 21 yrs old admitted to hospitals in Michigan in the 2002/2003 season found 8 cases of influenza-associated encephalopathy (including 2 deaths), Japan had previously reported similar complications. Reye’s syndrome – the effects on the liver (fatty deposits) and brain (edema) are particularly serious, get vomiting, lethargy, may develop coma. Reye’s syndrome is rare, but mortality rate may be as high as 40% of cases. The origin of the syndrome is unclear but it seems to follow certain viral infections such as influenza or chicken pox (varicella zoster/herpes zoster), especially in the young and especially if they have been treated with aspirin. Aspirin is contraindicated for childhood or adolescent fevers because it is a risk factor in the development of Reye's syndrome. Acetaminophen and ibuprofen are apparently not associated with Reye's syndrome. Guillain-Barré syndrome – targets peripheral nervous system - the cause of this is mysterious, recent vaccines do not seem to increase the risk of developing this. Major causes of influenza-associated death are bacterial pneumonia and cardiac failure. 90% of deaths are in people over 65 yrs of age. DIAGNOSIS Firm diagnosis is by means of virus isolation, and/or serology. Virus can be isolated from nose or throat swabs. Virus is grown in cultured cells or eggs. Hemadsorption may be used to detect infected cells. PCR (polymerase chain reaction) tests are being developed to detect viral RNA. Recently rapid tests which can be used in the physician’s office have been approved. Provisional diagnosis is often made clinically based on knowledge of a current outbreak of influenza combined with appropriate clinical symptoms (fever, cough, runny nose, malaise). EPIDEMIOLOGY HA (hemagglutinin) protein: Involved in attachment, as well as in membrane fusion in the endosomes. The receptor binding site is in a pocket and not exposed to the immune system. The antigenic domains are on the surface, if these are altered influenza virus can avoid the humoral response without affecting its ability to bind to the receptor.
NA (neuraminidase) protein: Neuraminidase digests sialic acid/ neuraminic acid â€“ which most cells have on their cell surface. Sialic acid is part of the virus receptor. If the virus binds to the cell, it will be endocytosed before the sialic acid is removed. However, by late in infection, the sialic acid will have been removed from the infected cell surface, and so it is easier for the progeny virions to diffuse away once they exit the cell. Neuraminidase is also involved in penetration of the mucus layer in the respiratory tract. Antigenic drift (due to mutation): Antibodies to HA are most important in protection, although those to NA also play a role. Both proteins undergo antigenic drift (i.e. accumulate mutations) and may accumulate changes such that an individual immune to the original strain is not immune to the drifted one. Antigenic drift results in sporadic outbreaks and limited epidemics. Antigenic shift (due to reassortment): In the case of influenza A, periodically get shift â€“ virus has an apparently "new" HA and/or NA. Thus, there is little pre-existing immunity (particularly if HA and NA both change, or if new HA) and an epidemic/pandemic is seen.
Ryan et al., in Sherris Medical Microbiology
Where does the "new" HA and/or NA come from? All (16) HA and (9) NA types circulate in ducks, some also circulate in other animals. We think that some animal somewhere (possibly a pig) gets infected with both human and animal viruses and that in one of the reassortants a few of the human virus genes are replaced with the animal virus genes, including genes for a new HA and/or NA segment. If this reassortant virus can infect humans, it will have mainly the same internal components as the current human virus but new envelope components and so it can grow well in human cells but we will have little immunity. Influenza A subtypes are therefore classified according to the type of HA and NA protein. Classification of influenza strains: Type (A/B/C)/place isolated/number of isolate/year isolated In the case of influenza A also: HA subtype (H) and NA subtype (N) For example, the three strains for the 2008-2009 vaccine are: A/Brisbane/59/2007 (H1N1) A/Brisbane/10/2007 (H3N2) B/Florida/4/2006
(Note: there is concern about recent outbreaks of avian influenza due to a strain of H5N1 influenza A virus. This bird virus seems to be able to infect humans without having to undergo a recombination event in some other animal. The case fatality rate is high (~60%) in humans. Fortunately, as yet the virus does not readily spread from birds to humans or one human to another. However, there is concern that it might mutate, or undergo reassortment with a human influenza virus, and acquire the ability to spread rapidly from human to human while still being as virulent.) Possibly we don't see antigenic shift in influenza B because there is apparently no animal reservoir for this virus. SURVEILLANCE: A measure of the severity of influenza in any one year is the excess of deaths due to pneumonia or influenza compared to the seasonally adjusted norm. The World Health Organization (WHO) maintains constant surveillance of influenza outbreaks world wide and has a series of 'sentinel' labs to look for what is happening. The CDC does the same in this country and co-operates with WHO. Usually the most important influenza virus is influenza A, but in some seasons influenza B is the major cause of influenza. In recent years H1N1 and H3N2 have often co-circulated, the proportions of each can change dramatically from year to year.
actual percentage of deaths
56 CDC: http://www.cdc.gov/mmwr/pdf/rr/rr57e717.pdf
PREVENTION TWO TYPES OF VACCINE TRIVALENT INACTIVATED VACCINE (TIV): The trivalent inactivated vaccine (TIV) is an inactivated preparation of egg grown virus and is given by injection. Only certain formulations of the vaccine are FDA certified for young children â€“ the annual ACIP recommendations (see below) give details. Protection is via IgG antibodies. LIVE, ATTENUATED INFLUENZA VIRUS VACCINE (LAIV) The live, attenuated influenza virus (LAIV) vaccine (see Genetics Lecture # 64) is prepared from egg-grown virus. It is approved for healthy (those not at risk for complications from influenza infection), non-pregnant individuals 2-49 years old but should not be given to children under 5yrs
of age who have possible reactive airways disease (for example, a history of recurrent wheezing). It is given nasally and should provide mucosal, humoral and cell-mediated immunity. It is contraindicated for children and adolescents on any therapy containing aspirin due to the potential risk of Reye’s syndrome since the virus is a live virus. Both influenza vaccines are formulated annually using the types and strains of influenza predicted to be the major problems for that year (the predictions are based on worldwide monitoring of influenza). The vaccines are multivalent, the current ones are trivalent and have two strains of influenza A and one of influenza B. Vaccination needs to be given every year because the most effective strains for the vaccine will change due to drift and/or shift. The vaccines are usually given in the Fall - once the strains to be used for the influenza season have been determined in the earlier part of the year, it takes time for the vaccine to be prepared. By giving the vaccine in the fall, protection should be high at the time the influenza season peaks. Both vaccines are grown in eggs and contraindicated for those allergic to eggs. The Advisory Committee on Immunization Practices (or ACIP) Recommendations for the Prevention and Control of Influenza is published annually by CDC in the Morbidity and Mortality Weekly Report (MMWR), Recommendations and Reports (MMWR-RR). This is available at http://www.cdc.gov/mmwr/mmwr_rr.html (note that the space is an underline character) - this page lists all recommendations and reports. This year’s influenza report is at (http://www.cdc.gov/mmwr/PDF/rr/rr5707.pdf ). This 64 page document reviews influenza, the detailed rationale about who should or should not receive vaccine, when it should be administered, and antiviral drugs. A brief summary of the recommendations from CDC follows, for more detailed information see the ACIP MMWR-RR for the current year. Annual vaccination against influenza for adults: Annual vaccination against influenza is recommended for any adult who wants to reduce the risk for becoming ill with influenza or of transmitting it to others. Vaccination also is recommended for all adults in the following groups, because these persons are either at high risk for influenza complications, or are close contacts of persons at higher risk: • persons aged >50 years (from public health point of view, easier to target by age than by high-risk condition, which may not have been discovered) • women who will be pregnant during the influenza season • persons who have chronic pulmonary (including asthma), cardiovascular (except hypertension), renal, hepatic, hematological or metabolic disorders (including diabetes mellitus) • persons who have immunosuppression (including immunosuppression caused by medications or by human immunodeficiency virus) • persons who have any condition (e.g., cognitive dysfunction, spinal cord injuries, seizure disorders, or other neuromuscular disorders) that can compromise respiratory function or the handling of respiratory secretions or that can increase the risk for aspiration • residents of nursing homes and other chronic-care facilities • health-care personnel • household contacts and caregivers of children aged <5 years and adults aged >50 years, with particular emphasis on vaccinating contacts of children aged <6 months • household contacts and caregivers of persons with medical conditions that put them at high risk for severe complications from influenza
Annual vaccination against influenza for children and adolescents (6mths -18 years) Vaccination of all children aged 6 months–18 years should begin before or during the 2008–09 influenza season if feasible, but no later than during the 2009–10 influenza season. Children and adolescents at high risk for influenza complications should continue to be a focus of vaccination efforts as providers and programs transition to routinely vaccinating all children and adolescents. Children and adolescents at higher risk for influenza complication are those: • aged 6 months–4 years • who have chronic pulmonary (including asthma), cardiovascular (except hypertension), renal, hepatic, hematological or metabolic disorders (including diabetes mellitus) • who are immunosuppressed (including immunosuppression caused by medications or by human immunodeficiency virus) • who have any condition (e.g., cognitive dysfunction, spinal cord injuries, seizure disorders, or other neuromuscular disorders) that can compromise respiratory function or the handling of respiratory secretions or that can increase the risk for aspiration • who are receiving long-term aspirin therapy who therefore might be at risk for experiencing Reye syndrome after influenza virus infection • who are residents of chronic-care facilities • who will be pregnant during the influenza season
PERSONS WHO SHOULD NOT BE VACCINATED WITH EITHER VACCINE: Vaccine should not be administered to persons known to have anaphylactic hypersensitivity to eggs or to other components of either inactivated or LAIV influenza vaccine without first consulting a physician. Prophylactic use of antiviral agents is an option for preventing influenza among such persons. See CDC documentation for further details on latest recommendations. USE OF ANTIVIRAL AGENTS IN PREVENTION AND TREATMENT Two neuraminidase inhibitors are currently available (zanamivir [Relenza] and oseltamivir [Tamiflu]). They are active against influenza A and influenza B. Both drugs are approved for prevention (about 70-90% effective in healthy adults). These drugs can also reduce the duration of uncomplicated influenza (by ~ 1day) and decrease symptoms if taken within 2 days of the onset of illness. To date there are only a few studies of how effective these drugs are in reducing serious complications in high risk groups when used to treat influenza (as contrasted with when used prophylactically). Some limited data suggest they may be beneficial. Rimantadine and amantadine block virus entry across the endosome, and also interfere with virus release. These drugs were widely used. However, in the 2005-2006 influenza season 92% of the H3N2 strains examined had a mutation which would confer resistance to these drugs as did 25% of the H1N1 strains tested - similar problems have been seen in seasons since then so these drugs are not recommended until the % resistance in the major circulating strains drops.In the absence of the resistant mutations they were good prophylactic agents for influenza A (but not for influenza B), although there are some problems in taking them on a long term basis. They could be given to protect during an outbreak - especially those at severe risk and key personnel. They could also be given at the time of vaccination for a few weeks - until the humoral response had time to develop. There is some evidence that rimantidine and amantadine can reduce the duration of non-resistant influenza A if given early in infection. Check with the CDC MMWR Recommendations and Reports for Influenza for concerns such as latest information on drug efficacy, dosage, side effects, and the annual update of recommendations.
Other treatment: Rest, liquids, anti-febrile agents (not aspirin in the young or adolescent, since Reyeâ€™s syndrome is a potential problem). Be aware of and treat complications appropriately.
TYPE A Severity of illness Animal reservoir Human pandemics Human epidemics Antigenic changes segmented genome Amantadine, rimantidine Zanamivir, Oseltamivir surface glycoproteins
++++ ++ yes no yes no yes yes shift, drift drift yes yes sensitive no effect (but current strains resistant) sensitive sensitive 2 2
TYPE C + no no no(sporadic) drift yes no effect