Each year I teach basic virology to medical, dental, and nursing students here at Columbia University Medical Center. You can find all the lecture videos, slides, and readings here at virology blog: virology.ws/course.
pathogenesis
Influenza 101
Soon after the new influenza H1N1 strain emerged in April 2009, I began a series of blog posts on basic aspects of influenza virus replication and pathogenesis. The goal of this series is to provide information that will allow everyone to better understand the events surrounding emergence and spread of the new pandemic strain.
Unfortunately blog posts tend to become invisible after a certain period of time, which does not befit educational material. Therefore I have made a list of these articles, with links, to make it easier for everyone to take Influenza 101. As new basic information on influenza is added to the blog, it will appear on the Influenza 101 page.
You can find Influenza 101 by clicking the tab above the banner image of this blog, or by going to this page.
Class is still in session.
Innate immune defenses
In response to viral infection, many organisms mount a remarkable defense known as the immune response. This response to viral infection consists of an innate, or nonspecific component, and an adaptive, or specific defense. The innate response is considered the first line of immune defense because it is active even before infection begins. In fact, many viral infections are halted by the innate immune system, which responds very quickly – within minutes to hours after infection.
A key property of the innate immune system is the ability to recognize viruses as ‘foreign’. Viral proteins and nucleic acids are distinguished from cellular counterparts by cellular proteins called pattern recognition receptors (illustrated). These are proteins present either in the cell cytoplasm or on cellular membranes, where they detect viral components. For example, the cytoplasmic protein RIG-I detects double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) with a 5′-triphosphate. These types of RNAs are usually not found in the cytoplasm of unifected cells; rather they are typically products of viral replication. When RIG-I binds these viral RNAs, a series of reactions occur which lead to the synthesis of cytokines, the primary output of the innate defense system. Other detectors of viruses are the membrane-bound toll-like receptors (TLRs), which sense viral glycoproteins, dsRNA, ssRNA, and the sequence CpG in viral DNA. Engagement of TLRs by these virus-specific ligands also leads to the synthesis of cytokines, albeit by different pathways.
The presence of cytokines in the blood is typically one of the earliest indications that the host has been infected with a virus. Over 80 known cytokines are secreted by infected cells. The first that are produced after viral infection include interferon-α and -β (IFN-α, IFN-β), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-12, and IFN-γ.
Cytokines function locally by binding receptors on other cells. For example, IFN produced by infected cells engages receptors on neighboring cells. Those cells then produce hundreds of cellular proteins which have antiviral activities. When cytokines enter the circulation, they elicit symptoms typical of many viral infections, including fever, sleepiness, lethargy, muscle pain, loss of appetite, and nausea.
Another key component of the innate response are the so-called sentinel cells: dendritic cells and macrophages present in peripheral compartments such as skin and mucosal surfaces. Sentinel cells patrol the body, seeking signs of infection. Dendritic cells bind cytokines produced by virus-infected cells, and also take up viral proteins released from dying virus-infected cells. They respond by producing more cytokines to amplify the original response.
In many viral infections, the early action of cytokines produced by infected cells and dendritic cells is sufficient to eliminate the pathogen. If innate defenses are overwhelmed and virus replication continues unabated, then the second-line defenses are mobilized to ensure host survival. These comprise the adaptive immune response – antibodies and immune cells. Days to weeks are required to mount an adaptive immune response that is specifically tailored to the infecting virus. The innate response therefore serves as a crucial rapid response that provides sufficient time for the activation of the adaptive immune system.
Pathogenesis of influenza in humans
When influenza virus is introduced into the respiratory tract, by aerosol or by contact with saliva or other respiratory secretions from an infected individual, it attaches to and replicates in epithelial cells. The virus replicates in cells of both the upper and lower respiratory tract. Viral replication combined with the immune response to infection (which we’ll discuss in later posts) lead to destruction and loss of cells lining the respiratory tract. As infection subsides, the epithelium is regenerated, a process that can take up to a month. Cough and weakness may persist for up to 2 weeks after infection.
A recent paper compiled data from a number of studies in which human volunteers were given influenza virus, and the production of virus and flu-like symptoms were recorded. The results are summarized in this graph:
Volunteers were infected with influenza virus by intranasal instillation, and virus titers were determined in daily nasal washes. Symptoms monitored included nasal stuffiness, runny nose, sore throat, sneezing, hoarseness, ear pressure, earache, cough, breathing difficulty, chest discomfort, and fever. The results indicate that viral shedding precedes illness by one day, but the curves are otherwise very similar. Most infections are mild and complete in 5 days but some continued for a week. Interestingly, 1 in 3 volunteers did not develop clinical illness but nevertheless shed virus.
These experimental findings most likely do not completely duplicate what occurs in natural influenza infections. First, the study did not involve children or older individuals, in whom the disease course is likely to be different. Furthermore, the pattern of infection will vary depending on the strain of influenza and the immunological status of the host.
Influenza complications of the upper and lower respiratory tract are common. These include otitis media, sinusitis, bronchitis, and croup. Pneumonia is among the more severe complications of influenza infection, an event most frequently observed in children or adults. In primary viral pneumonia, the virus replicates in alveolar epithelial cells, leading to rupture of walls of alveoli and bronchioles. Influenza H5N1 viruses frequently cause primary viral pneumonia characterized by diffuse alveolar damage and interstitial fibrosis. Primary viral pneumonia occurs mostly in individuals at high risk for influenza complications (e.g. elderly patients) but a quarter of the cases occur in those not at risk, including pregnant women.
Combined viral-bacterial pneumonia is common. In secondary bacterial pneumonia, the patient appears to be recovering from uncomplicated influenza but then develops shaking chills, pleuritic chest pain, and coughs up bloody or purulent sputum. Often influenza virus can no longer be isolated from such cases. The most common bacteria causing influenza associated pneumonia are Streptococcus pneumoniae, Staphylococcus aureus, and Hemophilus influenzae. These cases can be treated with antibiotics but the case fatality rate is still about 7%. Secondary bacterial pneumonia was a major cause of death during the 1918-19 influenza pandemic, during which antibiotics were not available.
The reasons why influenza virus infections may lead to pneumonia are not understood. Several hypotheses have been proposed and disproved over the years, including one in which reduced numbers of lymphocytes allow increased susceptibility to superinfection.
Carrat, F., Vergu, E., Ferguson, N., Lemaitre, M., Cauchemez, S., Leach, S., & Valleron, A. (2008). Time Lines of Infection and Disease in Human Influenza: A Review of Volunteer Challenge Studies American Journal of Epidemiology, 167 (7), 775-785 DOI: 10.1093/aje/kwm375
Stegemann, S., Dahlberg, S., Kröger, A., Gereke, M., Bruder, D., Henriques-Normark, B., & Gunzer, M. (2009). Increased Susceptibility for Superinfection with Streptococcus pneumoniae during Influenza Virus Infection Is Not Caused by TLR7-Mediated Lymphopenia PLoS ONE, 4 (3) DOI: 10.1371/journal.pone.0004840
Viruses and the respiratory tract
Now that we have a rudimentary understanding of influenza virus replication, we can begin to consider how the virus causes disease – a field of study called viral pathogenesis. The first step in this process is virus entry into the body.
The human body is covered with skin, which has a dead outer layer that cannot support viral replication and also serves as a impermeable barrier. Viruses may breach the skin via a vector bite, needle injury, animal bite, or abrasion. However, layers of exposed living cells must be present to absorb food, exchange gases, and release urine and other fluids. These mucosal layers serve as easy sites of entry for viruses.
The respiratory tract is the most common route of viral entry, a consequence of the exposed mucosal surface and the resting ventilation rate of 6 liters of air per minute. The huge absorptive area of the human lung (140 square meters) also plays a role. Large numbers of foreign particles and aerosolized droplets – often containing virions – are introduced into the respiratory tract each minute. The reason why we are not more frequently infected is that there are numerous defense mechanisms to protect the respiratory tract. Mechanical barriers abound – for example, the tract is lined with a mucociliary blanket comprising ciliated cells, mucus-secreting goblet cells, and subepithelial mucus-secreting glands. Foreign particles that enter the nasal cavity or upper respiratory tract are trapped in mucus and carried to the back of the throat, where they are swallowed. If particles reach the lower respiratory tract, they may also be trapped in mucus, which is then brought up and out of the lungs by ciliary action. The lowest reaches of the respiratory tract – the aveoli – are devoid of cilia. However, these gas-exchanging sacs are endowed with macrophages, whose job it is to ingest and destroy particles.
As we discussed previously, viruses may enter the respiratory tract in aerosolized droplets produced by coughing, sneezing, or simply talking, singing, or breathing. Infection may also be spread by contact with saliva or other respiratory secretions from an infected individual. The larger aerosol droplets land in the nose, while smaller ones may venture deeper into the respiratory tract, even as far as the aveoli.
For a virus to successfully establish an infection in the respiratory tract, it must avoid being swept away by mucus or engulfed by alveolar macrophages. Then there are the more specific immune mechanisms that may intervene – a topic we’ll consider later. Suffice it to say that if a virus establishes an infection in the respiratory tract, it has surmounted a number of formidable barriers which ensure that we are not continuously infected.