What is Influenza (Flu)?

Flu is a contagious respiratory illness caused by influenza viruses that infect the nose, throat, and sometimes the lungs. It can cause mild to severe illness, and at times can lead to death. The best way to prevent flu is by getting a flu vaccine each year.

Flu Symptoms

Influenza (flu) can cause mild to severe illness, and at times can lead to death. Flu symptoms usually come on suddenly. People who have flu often feel some or all of these symptoms:

• fever* or feeling feverish/chills
• cough
• sore throat
• runny or stuffy nose
• muscle or body aches
• headaches
• fatigue (tiredness)
• some people may have vomiting and diarrhea, though this is more common in children than adults.

*It’s important to note that not everyone with flu will have a fever.

How Flu Spreads

Most experts believe that flu viruses spread mainly by tiny droplets made when people with flu cough, sneeze or talk. These droplets can land in the mouths or noses of people who are nearby. Less often, a person might get flu by touching a surface or object that has flu virus on it and then touching their own mouth, nose or possibly their eyes.

How Many People Get Sick with Flu Every Year?

A 2018 CDC study published in Clinical Infectious Diseases looked at the percentage of the U.S. population who were sickened by flu using two different methods and compared the findings. Both methods had similar findings, which suggested that on average, about 8% of the U.S. population gets sick from flu each season, with a range of between 3% and 11%, depending on the season.

Why is the 3% to 11% estimate different from the previously cited 5% to 20% range?

The commonly cited 5% to 20% estimate was based on a study that examined both symptomatic and asymptomatic influenza illness, which means it also looked at people who may have had the flu but never knew it because they didn’t have any symptoms. The 3% to 11% range is an estimate of the proportion of people who have symptomatic flu illness.

Who is most likely to be infected with influenza?

The same CID study found that children are most likely to get sick from flu and that people 65 and older are least likely to get sick from influenza. Median incidence values (or attack rate) by age group were 9.3% for children 0-17 years, 8.8% for adults 18-64 years, and 3.9% for adults 65 years and older. This means that children younger than 18 are more than twice as likely to develop a symptomatic flu infection than adults 65 and older.

How is seasonal incidence of influenza estimated?

Influenza virus infection is so common that the number of people infected each season can only be estimated. These statistical estimations are based on CDC-measured flu hospitalization rates that are adjusted to produce an estimate of the total number of influenza infections in the United States for a given flu season.

The estimates for the number of infections are then divided by the census population to estimate the seasonal incidence (or attack rate) of influenza.

Does seasonal incidence of influenza change based on the severity of flu season?

Yes. The proportion of people who get sick from flu varies. A paper published in CID found that between 3% and 11% of the U.S. population gets infected and develops flu symptoms each year. The 3% estimate is from the 2011-2012 season, which was an H1N1-predominant season classified as being of low severity. The estimated incidence of flu illness during two seasons was around 11%; 2012-2013 was an H3N2-predominant season classified as being of moderate severity, while 2014-2015 was an H3N2 predominant season classified as being of high severity.

Table 1. Estimates of the Incidence of Symptomatic Influenza by Season and Age-Group, United States, 2010–2016

Period of Contagiousness

You may be able to spread flu to someone else before you know you are sick, as well as while you are sick.

• People with flu are most contagious in the first 3-4 days after their illness begins.
• Some otherwise healthy adults may be able to infect others beginning 1 day before symptoms develop and up to 5 to 7 days after becoming sick.
• Some people, especially young children and people with weakened immune systems, might be able to infect others for an even longer time.

Onset of Symptoms

The time from when a person is exposed and infected with flu to when symptoms begin is about 2 days, but can range from about 1 to 4 days.

Complications of Flu

Complications of flu can include bacterial pneumonia, ear infections, sinus infections and worsening of chronic medical conditions, such as congestive heart failure, asthma, or diabetes.

People at High Risk from Flu

Anyone can get flu (even healthy people), and serious problems related to flu can happen at any age, but some people are at high risk of developing serious flu-related complications if they get sick. This includes people 65 years and older, people of any age with certain chronic medical conditions (such as asthma, diabetes, or heart disease), pregnant women, and children younger than 5 years.

Preventing Seasonal Flu

The first and most important step in preventing flu is to get a flu vaccine each year. Flu vaccine has been shown to reduce flu related illnesses and the risk of serious flu complications that can result in hospitalization or even death. CDC also recommends everyday preventive actions (like staying away from people who are sick, covering coughs and sneezes and frequent handwashing) to help slow the spread of germs that cause respiratory (nose, throat, and lungs) illnesses, like flu.

Diagnosing Flu

It is very difficult to distinguish flu from other viral or bacterial respiratory illnesses based on symptoms alone. There are tests available to diagnose flu.

Treating Flu

There are influenza antiviral drugs that can be used to treat flu illness.

There are four types of influenza viruses: A, B, C and D. Human influenza A and B viruses cause seasonal epidemics of disease (known as flu season) almost every winter in the United States. Influenza A viruses are the only influenza viruses known to cause flu pandemics, i.e., global epidemics of flu disease. A pandemic can occur when a new and different influenza A virus emerges that both infects people and has the ability to spread efficiently among people. Influenza C virus infections generally cause mild illness and are not thought to cause human epidemics. Influenza D viruses primarily affect cattle and are not known to infect or cause illness in people.

Influenza A viruses are divided into subtypes based on two proteins on the surface of the virus: hemagglutinin (H) and neuraminidase (N). There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes (H1 through H18 and N1 through N11, respectively). While more than 130 influenza A subtype combinations have been identified in nature, primarily from wild birds, there are potentially many more influenza A subtype combinations given the propensity for virus “reassortment.” Reassortment is a process by which influenza viruses swap gene segments. Reassortment can occur when two influenza viruses infect a host at the same time and swap genetic information. Current subtypes of influenza A viruses that routinely circulate in people include: A(H1N1) and A(H3N2). Influenza A subtypes can be further broken down into different genetic “clades” and “sub-clades.” See the “Influenza Viruses” graphic below for a visual depiction of these classifications.

Clades and sub-clades can be alternatively called “groups” and “sub-groups,” respectively. An influenza clade or group is a further subdivision of influenza viruses (beyond subtypes or lineages) based on the similarity of their HA gene sequences. (See the Genome Sequencing and Genetic Characterization page for more information). Clades and subclades are shown on phylogenetic trees as groups of viruses that usually have similar genetic changes (i.e., nucleotide or amino acid changes) and have a single common ancestor represented as a node in the tree (see Figure 1). Dividing viruses into clades and subclades allows flu experts to track the proportion of viruses from different clades in circulation.

Note that clades and sub-clades that are genetically different from others are not necessarily antigenically different. This is best understood by first introducing the concepts of “antigens” and “antigenic properties.” As previously described, flu viruses have hemagglutinin (H) and neuraminidase (N) surface proteins. These proteins act as antigens. Antigens are molecular structures on the surface of viruses that are recognized by the immune system and can trigger an immune response (such as antibody production). The “antigenic properties” are a reflection of the antibody or immune response triggered by the antigens on a particular virus. When two flu viruses are antigenically different, this means that a host’s immune response (antibodies) elicited by infection or vaccination with one of the viruses will not as easily recognize and neutralize the other virus. Therefore, for antigenically different viruses, immunity developed against one of the viruses will not necessarily protect against the other virus as well.

Conversely, when two flu viruses are antigenically similar, a host’s immune response (antibodies) elicited by infection or vaccination with one of the viruses will recognize and neutralize the other virus, thereby protecting against the other virus.

Currently circulating influenza A(H1N1) viruses are related to the pandemic 2009 H1N1 virus that emerged in the spring of 2009 and caused a flu pandemic (CDC 2009 H1N1 Flu website). These viruses, scientifically called the “A(H1N1)pdm09 virus,” and more generally called “2009 H1N1,” have continued to circulate seasonally since then and have undergone genetic changes and changes to their antigenic properties (i.e., the properties of the virus that affect immunity).

Influenza A(H3N2) viruses also change both genetically and antigenically. Influenza A(H3N2) viruses have formed many separate, genetically different clades in recent years that continue to co-circulate.

Influenza B viruses are not divided into subtypes, but instead are further classified into two lineages: B/Yamagata and B/Victoria. Similar to influenza A viruses, influenza B viruses can then be further classified into specific clades and sub-clades. Influenza B viruses generally change more slowly in terms of their genetic and antigenic properties than influenza A viruses, especially influenza A(H3N2) viruses. Influenza surveillance data from recent years shows co-circulation of influenza B viruses from both lineages in the United States and around the world. However, the proportion of influenza B viruses from each lineage that circulate can vary by geographic location and by season. In recent years, flu B/Yamagata viruses have circulated much less frequently in comparison to flu B/Victoria viruses globally.

Naming Influenza Viruses

CDC follows an internationally accepted naming convention for influenza viruses. This convention was accepted by WHO in 1979 and published in February 1980 in the Bulletin of the World Health Organization, 58(4):585-591 (1980) (see A revision of the system of nomenclature for influenza viruses: a WHO Memorandum pdf icon[854 KB, 7 pages]external icon). The approach uses the following components:

• The antigenic type (e.g., A, B, C, D)
• The host of origin (e.g., swine, equine, chicken, etc.). For human-origin viruses, no host of origin designation is given. Note the following examples:
  • (Duck example): avian influenza A(H1N1), A/duck/Alberta/35/76
  • (Human example): seasonal influenza A(H3N2), A/Perth/16/2019
• Geographical origin (e.g., Denver, Taiwan, etc.)
• Strain number (e.g., 7, 15, etc.)
• Year of collection (e.g., 57, 2009, etc.)
• For influenza A viruses, the hemagglutinin and neuraminidase antigen description are provided in parentheses (e.g., influenza A(H1N1) virus, influenza A(H5N1) virus)
• The 2009 pandemic virus was assigned a distinct name: A(H1N1)pdm09 to distinguish it from the seasonal influenza A(H1N1) viruses that circulated prior to the pandemic.
• When humans are infected with influenza viruses that normally circulate in swine (pigs), these viruses are call variant viruses and are designated with the letter “v” (e.g., an A(H3N2)v virus).

Influenza Vaccine Viruses

Seasonal flu vaccines are formulated to protect against influenza viruses known to cause epidemics, including: one influenza A(H1N1) virus, one influenza A(H3N2) virus, one influenza B/Victoria lineage virus, and one influenza B/Yamagata lineage virus. Getting a flu vaccine can protect against these viruses as well as additional flu viruses that are antigenically similar to the viruses used to make the vaccine. Information about this season’s vaccine can be found at Preventing Seasonal Flu with Vaccination. Seasonal flu vaccines do not protect against influenza C or D viruses or against zoonotic (animal-origin) flu viruses that can cause human infections, such as variant or avian flu viruses. In addition, flu vaccines will NOT protect against infection and illness caused by other viruses that also can cause influenza-like symptoms. There are many other viruses besides influenza that can result in influenza-like illness (ILI) that spread during flu season.

Influenza (flu) viruses are constantly changing. They can change in two different ways.

Antigenic Drift

One way flu viruses change is called “antigenic drift.” Drift consists of small changes (or mutations) in the genes of influenza viruses that can lead to changes in the surface proteins of the virus, HA (hemagglutinin) and NA (neuraminidase). The HA and NA surface proteins of influenza viruses are “antigens,” which means they are recognized by the immune system and are capable of triggering an immune response, including production of antibodies that can block infection. The changes associated with antigenic drift happen continually over time as flu viruses replicate (i.e., infect a host and make copies of themselves). Most flu shots are designed to target the HA surface proteins/antigens of flu viruses. The nasal spray flu vaccine (LAIV) may target both the HA and NA of a flu virus.

The small changes that occur from antigenic drift usually produce viruses that are closely related to one another, which can be illustrated by their location close together on a phylogenetic tree. Flu viruses that are closely related to each other usually have similar antigenic properties. This means that antibodies your immune system creates against one flu virus will likely recognize and respond to antigenically similar flu viruses (this is called “cross-protection”).

However, the small changes associated with antigenic drift can accumulate over time and result in viruses that are antigenically different (further away on the phylogenetic tree). It also is possible for a single change in a particularly important location on the HA to result in antigenic drift. When antigenic drift occurs, the body’s immune system may not recognize and prevent sickness caused by the newer flu viruses. As a result, a person becomes susceptible to flu infection again, as antigenic drift has changed the virus’ antigenic properties enough that a person’s existing antibodies won’t recognize and neutralize the newer flu viruses.

Antigenic drift is an important reason why people can get flu more than one time. Drift is also a primary reason why the composition of flu vaccines for use in the Northern and Southern Hemispheres is reviewed annually and updated as needed to keep up with evolving flu viruses.

Antigenic Shift

Another type of change is called “antigenic shift.” Shift is an abrupt, major change in a flu A virus, resulting in new HA and/or new HA and NA proteins in flu viruses that infect humans. Antigenic shift can result in a new flu A subtype. Shift can happen if a flu virus from an animal population gains the ability to infect humans. Such animal-origin viruses can contain HA or HA/NA combinations that are different enough from human viruses that most people do not have immunity to the new (e.g., novel) virus. Such a “shift” occurred in the spring of 2009, when an H1N1 virus with genes from North American Swine, Eurasian Swine, humans and birds emerged to infect people and quickly spread, causing a pandemic. When shift happens, most people have little or no immunity against the new virus.

While flu viruses change all the time due to antigenic drift, antigenic shift happens less frequently. Flu pandemics occur rarely; there have been four flu pandemics in the past 100 years. For more information, see pandemic flu. Type A viruses undergo both antigenic drift and shift and are the only flu viruses known to cause pandemics, while flu type B viruses change only by the more gradual process of antigenic drift.

CDC Human Serology Efforts to Improve Seasonal Flu Vaccines and Prepare Against Future Flu Pandemics

To improve seasonal flu vaccines and prepare against future pandemics, CDC’s Influenza Division conducts a wide range of laboratory activities involving human serology. Serology is the scientific study of blood to look at the response of the immune system to vaccination or infections with pathogens, like flu viruses. CDC Influenza Division’s human serology work supports the following objectives:

• Improving assessment and selection of candidate vaccine viruses (CVVs) for use in producing seasonal flu vaccines.
• Increasing scientific understanding of the characteristics of the immune response following flu vaccination that are associated with immune protection (also known as “correlates of protection.”)
• Determining population immunity against circulating human seasonal flu viruses.
• Identifying asymptomatic or mild flu infections and improving the assessment of flu disease burden.
• Determining population immunity against zoonotic (animal-origin) flu viruses with pandemic potential.
• Improving the assessment and selection of CVVs for use in producing pre-pandemic flu vaccines

Human serology work to improve assessment and selection of CVVs for producing seasonal flu vaccines

CDC’s Influenza Division now uses human serology data to improve the selection of candidate vaccine viruses (CVVs) for use in producing seasonal flu vaccines. Flu viruses are always changing and many different flu viruses of various types and subtypes circulate around the world and cause seasonal flu epidemics. All flu viruses undergo small genetic changes over time, and these genetic changes can result in antigenic changes that allow a flu virus to evade a person’s existing immunity from prior flu infection or vaccination. More information is available at How the Flu Virus Can Change. This is one reason why the composition of flu vaccines must be reviewed and updated and new vaccines produced prior to each flu season.

The selection of CVVs for producing seasonal flu vaccines is made months prior to the start of the flu season in the Southern and Northern Hemispheres. Twice a year, the World Health Organization (WHO) organizes a consultation with the seven WHO Collaborating Centers, Essential Regulatory Laboratories, and representatives from key national laboratories and academia to review available data and make recommendations on the composition of flu vaccines for the coming season. These WHO consultations are called vaccine composition meetings (VCM). VCM occurs in February to recommend the composition of Northern Hemisphere flu vaccines and in September to recommend the composition of Southern Hemisphere flu vaccines. For more information, visit Selecting Viruses for the Seasonal Flu Vaccine.

Seasonal flu vaccines are designed to include CVVs that protect against the three or four (depending on vaccine) major flu virus groups (known as types, subtypes, and lineages) that often co-circulate during flu seasons. All available flu vaccines in the United States are quadrivalent (four-component) flu vaccines this season. Quadrivalent flu vaccines contain a flu A(H1N1)pdm09 component, a flu A(H3N2) component, a flu B/Yamagata lineage component, and a flu B/Victoria lineage component. Trivalent (three-component) flu vaccines contain only one flu type B virus. Therefore, three or four candidate vaccine viruses (CVVs) are chosen for use in flu vaccine production. Research indicates if the flu virus representing each vaccine component needs to be updated with a new CVV.

Human serology data contributes to the selection of CVVs for flu vaccines.

Human serology data is now included in the overall body of data that CDC collects to inform WHO’s Northern and Southern Hemisphere flu vaccine composition recommendations. To obtain this serology data, CDC works with partner organizations to collect blood samples from people who received flu vaccines. The blood samples are collected during the flu season, and this work begins shortly after flu vaccines becomes available. Blood samples are collected from people prior to vaccination and then again about three weeks to four weeks post-vaccination to allow time for their bodies to develop antibodies. Antibodies are proteins made by a person’s immune system in response to infections or vaccination that can protect against future infections. Following vaccination, a person’s antibodies will rise over time and generally peak around 3-4 weeks post-vaccination. Antibody levels from vaccination can wane over time, generally over the course of several months post-vaccination before leveling off. Although antibodies wane, this does not mean they drop to zero.

For purposes of serologic testing and analysis, laboratory scientists will take the blood samples collected from people and prepare sera from them. Serum (its plural form is sera) is a fluid component of blood from which blood clotting elements are removed. Sera contain antibodies and are used in serology tests. Serology tests, also known as antibody tests, are conducted on a person’s blood serum to look for the presence of antibodies. CDC collects blood samples from people of all age groups and from different geographic areas both before and after flu vaccination. After this blood is collected, CDC prepares sera from these blood samples to enable analysis of antibodies against circulating flu viruses. Laboratory tests including the hemagglutination inhibition assay (HI test) and microneutralization assay, are commonly used for serology testing. The goal is to measure how well the antibodies elicited from flu vaccination can recognize and neutralize the flu viruses in circulation. If the antibodies produced from vaccination effectively neutralize circulating flu viruses, then it is likely that the current flu vaccine’s composition is suitable for protecting people during that flu season. This means that the flu vaccine’s composition does not need to be updated. In contrast, if the antibodies produced from vaccination do not effectively neutralize currently circulating flu viruses, then it is likely that one or more of the components of the flu vaccine will need to be updated. All of CDC’s human serology data collection and analysis is completed prior to the WHO VCM in order to contribute to the flu vaccine composition recommendations.

How human serology data differs from the ferret sera used traditionally

Scientists have traditionally used sera produced by ferrets inoculated with specific flu viruses to assess the similarity (match) or differences (mismatch) between the CVVs chosen for use in flu vaccines and the flu viruses that are in circulation. By testing antibodies found in human sera after flu vaccination, scientists can better determine whether the human immune response to the vaccine will sufficiently target and neutralize circulating flu viruses.

The human immune response to flu vaccination can be different to that of ferrets. Ferrets used for this kind of testing are naïve to flu, meaning that they have never been infected with a flu virus or been vaccinated against flu. In contrast, humans – particularly older children and adults – often have had previous flu vaccinations or flu infections. These prior flu infections or vaccinations are known to affect the way the human immune system responds to future flu vaccinations or flu virus infections. For example, sometimes only antibodies contained in human sera (as opposed to ferret sera) can detect the antigenic differences between flu viruses. Also, sometimes people may respond differently to flu vaccination based on their age, exposure history to flu, geographic location, immune status, and other host factors.

As a result, CDC collects and tests sera from vaccinated people of all ages (from infants 6 months of age to adults ≥ 65 years) and from different geographic locations, both in the United States and around the world during each flu season. The collection and use of human serology data are critical to ensure that the CVVs used in seasonal flu vaccines can provide sufficient protection against circulating flu viruses each flu season.

Timing of human serology data collection

CDC collects human serology data year-round to inform the recommendations for the composition of seasonal flu vaccines. For WHO vaccine recommendations, there is only a short timeframe each season after flu vaccines become available during which CDC can collect and analyze the serology data prior to the WHO VCM. The WHO VCM takes place in February for the Northern Hemisphere and in September for the Southern Hemisphere. Therefore, CDC must collect and analyze human serology data twice a year in advance of these meetings to support the flu vaccine composition recommendation for the coming season.

Human serology work to better understand the correlates of protection

One goal of CDC Influenza Division’s human serology work is to increase scientific understanding of the characteristics of the immune response following vaccination that are associated with immune protection, also known as the “correlates of protection.” As a part of this work, CDC Influenza Division conducts immunogenicity studies to compare the protective benefits provided by different flu vaccine platforms. For example, studies are conducted comparing standard egg-based flu vaccines to cell-based, recombinant, adjuvanted, and high dose flu vaccines that are currently licensed to use in the United States. CDC also compares the human antibody response across different age groups and in different populations, including those with different immune status (e.g., children, elderly, health care workers, pregnant women, and people with underlining conditions). This data helps CDC determine the most effective flu vaccination strategies for specific age groups or populations.

Researching the causes of flu vaccine breakthrough infections

Human serology is also useful for studying flu “breakthrough infections,” which are flu infections that occur in people who have been vaccinated. CDC researchers want to better understand the immunological factors that are associated with breakthrough infections. For example, scientists have traditionally measured the amount of antibodies that a person produces following flu vaccination by a laboratory test called the hemagglutination inhibition test (HI test). The HI test is used to determine how well antibodies developed against one flu virus prevent the binding of another flu virus to red blood cells. In doing so, the test can determine how antigenically similar the two viruses are to one another. HI test is often used as a surrogate of the neutralization test. More information about the HI test and how it is used to inform vaccine composition recommendations is available at Antigenic Characterization | CDC.

Certain thresholds of HI antibody titers have been shown to be associated with a reduction in the risk of flu infection (immune protection). A titer is a laboratory unit of measurement for a specific amount of antibody in a serum. HI titers are often used to evaluate how well a flu vaccine works. However, recent data show that other immunological markers (for example, antibodies to the stalk region of the flu virus’ hemagglutinin (HA) surface proteins and antibodies to the virus’ neuraminidase (NA) surface proteins) may also contribute to immune protection.

To better understand the causes of flu vaccine breakthrough infections, CDC scientists analyze the sera collected from people with “breakthrough” flu infections using a variety of additional laboratory tests. In addition to microneutralization assays, which measure antibodies that can neutralize an influenza virus’ ability to cause infection, there are assays to measure antibodies that work against the flu virus’ neuraminidase (NA) surface protein; assays to measure antibodies that target the stalk region of a flu virus’ hemagglutinin (HA) surface protein; and other assays needed to identify additional immunological markers that correlate with protective immunity.

Data gathered using these tests have the potential to provide critical information that will improve understanding of the underlying immunological factors contributing to vaccine breakthrough infections. This information can in turn be used to inform the most effective vaccination strategies.

Learning why vaccine effectiveness varies by flu season

Another area of human serology research involves understanding why flu vaccine effectiveness varies from season to season and differs between flu virus types and subtypes. Many factors can impact the effectiveness of flu vaccines in the population. One factor is the similarity (or match) between the characteristics of the flu vaccine and the flu viruses that are circulating and causing illness. For example, many flu vaccines are still produced in chicken eggs, and the egg-based vaccine manufacturing production process can introduce genetic changes in vaccine viruses that allow them to grow better in chicken eggs. Such changes, however, can alter the antigenic properties of viruses, making them less similar to the flu viruses in circulation. These antigenic differences can potentially reduce the flu vaccine’s effectiveness against circulating flu viruses. In addition, complex host immunological factors also can affect the protective immunity people receive from flu vaccination. For example, “immune priming” is a concept where the benefit a person gets from vaccination (or the robustness of a person’s immune response to vaccination) may be affected by previous flu vaccinations or infections that occurred earlier in that person’s life.

Determining population immunity against circulating human seasonal flu viruses

CDC Influenza Division also conducts human serology work to measure population immunity against circulating seasonal flu viruses. This is done using seroprevalence surveys. A seroprevalence survey is an investigation that uses serology tests (also known as antibody tests) to estimate the percentage of people in a population that have antibodies against a virus of interest, such as seasonal flu viruses circulating among people. The presence of these antibodies in blood is an indicator of previous infection or vaccination. Seroprevalence surveys can tell us how many people in a specific population have existing antibodies against a particular flu virus. The percentage of people in a population who have antibodies to a particular virus is called “seroprevalence.”

Alternatively, such surveys can help determine what proportion of people in the population lack antibodies to emerging flu viruses, and therefore, are susceptible to infection with these viruses. CDC collaborates with partner organizations to collect blood samples from thousands of people of all age groups. Sera are collected from people living in diverse geographic locations across the United States so that the populations sampled are representative of the U.S. population.

How assessments of population immunity help with improving flu vaccines

Seroprevalence surveys can help CDC determine if the population surveyed is vulnerable to emerging flu viruses. This enables flu experts to determine whether the flu vaccine should be updated to protect against these flu viruses for the upcoming flu season. Flu experts choose vaccine viruses that provide broad protection against a range of flu viruses of the same flu type, subtype, or lineage. More information is available at types of flu viruses.

Using serology to identify past evidence of flu infection.

Seroprevalence surveys are useful for identifying past mild or asymptomatic flu infections (i.e., infections without symptoms of illness) in people who are no longer shedding virus. Such infections are often missed by traditional diagnostic tests. Identifying these types of infections is especially helpful during investigations of outbreaks caused by novel viruses. In addition, by capturing both asymptomatic and symptomatic infections, human serology data can help health officials better estimate the true burden of disease to implement effective vaccination strategies in the population.

Determining population immunity against zoonotic flu viruses with pandemic potential

Just as CDC uses seroprevalence studies to assess population immunity against seasonal flu viruses, CDC also uses the same seroprevalence surveys to assess population immunity against zoonotic (animal-origin) flu viruses with pandemic potential. For example, CDC can test the human antibodies collected by seroprevalence surveys to determine if people of a particular age or from a particular geographic area are vulnerable to infection with flu viruses that originate from animals. Examples of flu viruses that originate from animals include avian flu (bird flu) viruses or swine flu viruses (which are called “variant” flu viruses when they infect people). When a brand-new virus from animals infects people (i.e., a novel virus), the risk of the virus causing a pandemic can be high if the virus is highly transmissible (i.e., spreads easily among people) and the human population has low or no pre-existing immunity against the virus. CDC closely monitors the emergence of novel flu viruses and assesses their pandemic risk using a tool called Influenza Risk Assessment Tool (IRAT). Population immunity is a critical factor in assessing the pandemic potential of emerging novel flu viruses.

Improving the assessment and selection of CVVs for use in producing pre-pandemic flu vaccines

By using human serology data to determine people’s susceptibility to zoonotic flu viruses with pandemic potential, CDC can prioritize the development of pre-pandemic vaccines that protect against these viruses. For pandemic preparedness, the U.S. government has established pandemic vaccine stockpiles. As new flu viruses with pandemic potential emerge, CDC scientists evaluate whether stockpiled pre-pandemic vaccines can offer protection against them. This work helps determine whether new pre-pandemic vaccines are needed, and it informs the selection of CVVs to protect against novel flu viruses with pandemic potential.

“Antigens” are molecular structures on the surface of viruses that are recognized by the immune system and are capable of triggering one kind of immune response known as antibody production. Two proteins (hemagglutinin and neuraminidase) on the surface of influenza viruses contain the major antigens targeted by antibodies (see Figure 1).

When someone is exposed to an influenza virus (either through infection or vaccination) their immune system makes specific antibodies against the antigens (surface proteins) on that particular influenza virus. The term “antigenic properties” is used to describe the antibody or immune response triggered by the antigens on a particular virus. “Antigenic characterization” refers to the analysis of a virus’ antigenic properties to help assess how related it is to another virus.

CDC antigenically characterizes about 2,000 influenza viruses during a typical flu season to monitor for changes in circulating viruses and to compare how similar these viruses are to those included in vaccines. Antigenic characterization can give an indication of the flu vaccine’s ability to produce an immune response against the influenza viruses circulating in people. This information also helps experts decide what viruses should be included in the upcoming season’s influenza vaccine.

Serology tests using human sera and genetic sequencing provide additional information about how similar circulating flu viruses are to vaccine viruses or other influenza viruses.

Figure 1. Influenza Virus Features

The above image shows the different features of an influenza virus, including the main surface proteins, hemagglutinin (HA) and neuraminidase (NA). Following influenza infection or receipt of a flu vaccine, the body’s immune system develops antibodies that recognize and bind to “antigenic sites,” which are regions found on an influenza virus’ surface proteins. By binding to the HA antigenic sites, antibodies can neutralize influenza viruses, which prevents them from causing further infection. (Antibodies that bind to the NA antigenic sites can also reduce further spread.)

The Hemagglutination Inhibition Assay (HI Test)

Scientists use a test called the hemagglutination inhibition assay (HI test) to antigenically characterize influenza viruses. HA proteins on the surface of influenza viruses can bind to red blood cells and “glue” them together, forming a lattice structure (this is known as “hemagglutination”). The picture below provides an example of hemagglutination. The rightmost section of the image shows what hemagglutination looks like in the well of a microtiter plate (this is explained in greater detail further below).

The HI test works by measuring how well antibodies bind to the HA proteins and prevent them from “gluing” red blood cells together (i.e., hemagglutination inhibition). The picture below provides an example of hemagglutination inhibition. The rightmost section of the image shows what hemagglutination inhibition looks like in the well of a microtiter plate (this is explained in greater detail further below).

Scientists use the HI test to assess the antigenic similarity between influenza viruses. This test helps to select the candidate vaccine viruses (CVVs) included in seasonal flu vaccines. HI test results can tell us whether antibodies developed after vaccination with one virus recognize, bind to, and therefore, are similar to other circulating influenza viruses. Scientists also use the HI test to compare the antigenic properties (i.e., the virus’ ability to be recognized by antibodies) of currently circulating influenza viruses with those of influenza viruses that have circulated in the past.

The HI test involves three main components: antibodies, influenza virus, and red blood cells that are mixed together in the wells (i.e., cups) of a microtiter plate. (See Image 1.)

Image 1. A Microtiter Plate

A microtiter plate is used to perform the HI test. The plate contains wells (i.e., cup-like depressions that can hold a small amount of liquid) where the solution of antibodies, influenza virus and red blood cells are inserted and allowed to interact. These wells are arranged according to rows and columns (which are identified on the microtiter plate by letters and numbers, respectively). The rows of the plate can be used to test different influenza viruses against the same set of antibodies. The columns can be used to differentiate between greater dilutions of antibodies, like a scale from low to high going from left to right (see Figures 3 and 4 for an example).

The antibodies used in the HI test are obtained by infecting an animal (usually a ferret) that is immunologically naïve (i.e., it has not been exposed to any influenza virus or vaccine previously in its lifetime). The animal’s immune system creates antibodies in response to the antigens on the surface of the specific flu virus that was used to infect that animal. To study these antibodies, a sample of blood is drawn from the animal, from which serum is obtained. The HI test measures how well these antibodies recognize and bind to other influenza viruses (such as, influenza viruses isolated from flu patients). If the ferret antibodies that resulted from exposure to the vaccine virus recognize and bind well to the influenza virus from a sick patient, this indicates that the vaccine virus is antigenically similar to the influenza virus obtained from the sick patient. This finding has implications for how well the vaccine might work in people. See Flu Vaccine Effectiveness: Questions and Answers for Health Professionals for more information.

As previously mentioned, the influenza viruses used in the HI test are obtained from sick people. CDC and other WHO collaborating centers collect specimens from people all over the world to track which influenza viruses are infecting humans and to monitor how these viruses are changing.

For the HI test, red blood cells (RBCs) are taken from animals (usually turkeys or guinea pigs). They are used in the HI test because influenza viruses bind to them. Normally, RBCs in a solution will sink to the bottom of the assay well and form a red dot at the bottom (Figure 2A). However, when an influenza virus is added to the RBC solution, the virus’ hemagglutinin (HA) surface proteins will bind to multiple RBCs. When influenza viruses bind to the RBCs, the red cells form a lattice structure (Figure 2B). This keeps the RBCs suspended in solution instead of sinking to the bottom and forming the red dot. The process of the influenza virus binding to RBCs to form the lattice structure is called “hemagglutination.”

Figure 2. Components of the HI Assay

The HI test involves the interaction of red blood cells (RBCs), antibody and influenza virus. Row A shows that in the absence of virus, RBCs in a solution will sink to the bottom of a microtiter plate well and look like a red dot. Row B shows that influenza viruses will bind to RBCs when placed in the same solution. This is called hemagglutination and is represented by the formation of the lattice structure, depicted in the far-right column under “Microtiter Results.” Row C shows that the antibody binds well to the virus and prevents the virus from gluing RBCs together and forming a lattice structure, therefore, hemagglutination does not occur (i.e., hemagglutination inhibition occurs instead).

When antibodies are pre-mixed with influenza virus followed by RBCs, the antibodies will bind to influenza virus antigens that they recognize, covering the virus so that its HA surface proteins can no longer bind to RBCs (Figure 2C). The reaction between the antibody and the virus inhibits (i.e., prevents) hemagglutination from occurring, which results in hemagglutination inhibition (as shown in Figure 2C). This is why the assay is called a “hemagglutination inhibition (HI) test.” Hemagglutination (as depicted in Figure 2B) occurs when antibodies do not recognize and bind to the influenza viruses in the solution, and as a result, the influenza viruses bind to the red blood cells, forming the lattice structure. When the antibodies do recognize and bind to the influenza virus in the solution, this shows that the vaccine virus (like the one the ferret was infected with) is similar to the influenza virus obtained from the sick patient. When this happens, the influenza virus being tested is said to be “antigenically similar” to the influenza virus that created the antibodies (from the ferret).

When a circulating influenza virus is antigenically different from a vaccine, the antibodies developed in response to the vaccine virus may not recognize and bind this virus. In the HI test, this will cause hemagglutination to occur (see Figure 2B). Circulating influenza viruses tested via the HI test are typically obtained from respiratory samples collected from sick patients.

Assessing Antigenic Similarity Using the HI Test

The HI test assesses the degree of antigenic similarity between two viruses using a scale based on antibody dilution. As previously mentioned, the HI test is performed using a microtiter plate. The microtiter plate contains rows and columns of wells (i.e., cups) where RBCs, influenza virus and antibodies (developed against a comparison virus, such as a vaccine virus) are mixed. Dilutions are marked across the top of the microtiter plate. These dilutions function as a scale for assessing antigenic similarity and immune response. By testing the ability of greater dilutions of antibody to prevent hemagglutination, scientists measure how well those antibodies recognize and bind to an influenza virus. The higher the dilution, the fewer antibodies are needed to block hemagglutination and the more antigenically similar the two viruses being compared are to each other. The highest dilution of antibody that results in hemagglutination inhibition of the test virus is considered an HI titer (Figure 3).

Figure 3. HI Titer Assay

This virus has an HI titer of 1280, which means that the greatest dilution of antibody that still blocked hemagglutination from occurring was at 1280 dilution. At this dilution, the antibodies were still capable of recognizing and binding to the antigens on the virus.

Figure 4. Antigenic Characterization Assay

When CDC antigenically characterizes influenza viruses to inform decisions on the formulation of the seasonal flu vaccine, the HI test is used to compare currently circulating viruses (rows B and C) with vaccine viruses (row A). This allows scientists to quickly determine if a virus used in the seasonal flu vaccine is antigenically similar to circulating influenza viruses and therefore capable of producing an immune response against them.

Public health experts consider influenza viruses to be antigenically similar or “like” each other if their HI titers differ by two dilutions or less. (This is equivalent to a two-well (i.e., a four-fold dilution) or less difference). Using Figure 4 as an example, when circulating virus 1 is compared to a vaccine virus, circulating virus 1 differs by one dilution (a 2-fold difference) and therefore is “like” the previous season’s vaccine virus. However, circulating virus 2 differs by five dilutions (a 32-fold difference) and therefore is not “like” the previous season’s vaccine virus. Circulating viruses that are antigenically dissimilar (i.e., not “like”) to vaccine virus are considered “low reactors.”

Limitations

Antigenic characterization gives important information about whether a vaccine made using a specific vaccine virus will protect against circulating influenza viruses, but there are several limitations to antigenic characterization test methodology, which are described below.

Does not work with recent Flu A(H3N2) viruses

An important limitation of the HI test is that the majority of circulating influenza A(H3N2) viruses do not bind to RBCs, and therefore, cannot be tested using the HI test. As a result, CDC has developed a different virus neutralization assay called the “high-content imaging-based neutralization test” (HINT) to measure how circulating A(H3N2) flu viruses are evolving to evade human immunity. More information about HINT is available in a CDC spotlight article.

Egg Adaptations

Right now, most flu vaccines are made using viruses grown in chicken eggs. As human influenza viruses adapt to grow in eggs, genetic changes can occur in the viruses. These are called “egg-adapted” changes. Some egg-adapted changes may change the virus’ antigenic (or immunogenic) properties while others may not. Egg-adapted changes have become a particular problem for selection of candidate vaccine viruses (CVVs) for the influenza A(H3N2) virus component of flu vaccine made using egg-based production technology. Influenza A(H3N2) viruses tend to grow less well in chicken eggs than other influenza viruses (e.g. A(H1N1)pdm09), and they also are more prone to egg-adapted changes. Such changes can reduce the immune protection provided by the flu vaccine against circulating A(H3N2) viruses.

Time from Vaccine Virus Selection to Delivery of Vaccine

Using current egg-based vaccine production technology, it takes about six months from the time that a vaccine virus is chosen (i.e., February for the Northern Hemisphere flu vaccine) to the time when influenza vaccines become widely available. Because influenza viruses are constantly changing, circulating influenza viruses may change during this six month period. If these genetic changes cause antigenic changes, it could mean that antibodies created through vaccination may not recognize and inactivate circulating influenza viruses. New technologies that shorten the manufacturing time could reduce the chances of significant antigenic changes occurring in circulating viruses before influenza vaccines become available each year: for example, cell-based and recombinant flu vaccines.

Use of Animals

Use of the HI test to assess the similarity of circulating influenza viruses to vaccine viruses involves obtaining antibodies from animals (particularly ferrets). Ferrets are often used as an animal model for influenza infection because they can be easily infected with human influenza viruses and experience similar signs and symptoms (e.g., fever, sneezing, loss of appetite, etc.). Prior to infection, ferret serum is pre-screened to determine that it does not contain antibodies to circulating influenza viruses. When ferrets are infected with a live influenza virus, they typically produce strong antibody responses against that particular virus. Therefore, ferret antisera are commonly used to detect antigenic differences between influenza viruses. However, there are differences between the immune responses of ferrets and humans which need to be considered when assessing the antigenic properties of influenza viruses.

Genome Sequencing

Influenza viruses are constantly changing, in fact all influenza viruses undergo genetic changes over time. An influenza virus’ genome consists of all genes that make up the virus. CDC conducts year-round surveillance of circulating influenza viruses to monitor changes in the genome of these viruses. This work is performed as part of routine U.S. influenza surveillance and as part of CDC’s role as a World Health Organization (WHO) Collaborating Center for the Surveillance, Epidemiology and Control of Influenza. The information CDC collects from studying genetic changes (also known as “substitutions” or “mutations”) in influenza viruses plays an important public health role by helping to determine whether vaccines and antiviral drugs will work against currently circulating influenza viruses, as well as helping to determine the potential for influenza viruses in animals to infect humans.

Genome sequencing is a process that determines the order, or sequence, of the nucleotides (i.e., A, C, G and T/U) in each of the genes present in the virus’s genome. Nucleotides are organic molecules that form the structural unit building block of nucleic acids, such as RNA and DNA. All influenza viruses consist of single-stranded RNA as opposed to dual-stranded DNA. The RNA genes of influenza viruses are made up of chains of nucleotides that are bonded together and coded by the letters A, C, G and U, which stand for adenine, cytosine, guanine, and uracil, respectively. Full genome sequencing can reveal the approximately 13,500-letter sequence of all the genes of the influenza virus’ genome.

The two influenza types (A and B) that cause seasonal epidemics have eight RNA gene segments. These genes contain “instructions” for making new viruses, thereby spreading infection. An influenza virus’s surface proteins, hemagglutinin (HA) and neuraminidase (NA), determine important properties of the virus and are included in most seasonal vaccines, which is why they are analyzed more closely. In a typical year, CDC performs whole genome sequencing on about 7,000 influenza viruses from original clinical samples collected through virologic surveillance.

Comparing the nucleotides in one gene of a virus with that of a different virus can reveal variations between the two viruses. Genetic variations are important because they can change amino acids that make up the influenza virus’s proteins, resulting in structural changes to the protein, and thereby altering properties of the virus. Some of these properties include the ability to evade human immunity, spread between people, and susceptibility to antiviral drugs. The changes to the proteins can come in the form of amino acid substitutions, insertions, or deletions.

Genome sequencing reveals the sequence of the nucleotides in a gene, like alphabet letters in words. Comparing the composition of nucleotides in one virus gene with the order of nucleotides in a different virus gene can reveal variations between the two viruses.

Genetic variations are important because they affect the structure of an influenza virus’ surface proteins. Proteins are made of sequences of amino acids.

The substitution of one amino acid for another can affect properties of a virus, such as how well a virus transmits between people, and how susceptible the virus is to antiviral drugs or current vaccines.

Genetic Characterization

CDC and other public health laboratories around the world have been sequencing the gene segments of influenza viruses since the 1980s. CDC contributes gene sequences to public databases, such as GenBank and the Global Initiative on Sharing Avian Influenza Data (GISAID), for use by researchers and public health scientists. The sequences deposited into these databases allow CDC and other researchers to compare the genes of currently circulating influenza viruses with the genes of older influenza viruses and those used in vaccines. This process of comparing genetic sequences is called genetic characterization. CDC uses genetic characterization for several reasons:

• To determine how closely “related” or similar flu viruses are to one another genetically
• To monitor how flu viruses are evolving or changing over time
• To identify genetic changes that affect the virus’s properties. For example, to identify the specific changes that are associated with influenza viruses spreading more easily, causing more severe disease, or developing resistance to antiviral drugs
• To assess how well a flu vaccine might protect against a particular influenza virus based on its genetic similarity to the virus
• To monitor for genetic changes in influenza viruses circulating in animal populations that could enable them to infect humans.

The genetic differences among a group of influenza viruses can be shown by organizing them into a graphic called a “phylogenetic tree.” Phylogenetic trees for influenza viruses are like family (genealogy) trees for people. These trees show how closely ‘related’ individual viruses are to one another. Each sequence from a specific influenza virus has its own branch on the tree. Viruses are grouped by comparing changes in nucleotides within the gene. Where branches meet, these “nodes” represent the common ancestor of the viruses and indicate that the viruses share similar genetic sequences. Viruses which share a common ancestor can also be described as belonging to the same clade. The degree of genetic difference (number of nucleotide differences) between viruses is represented by the length of the horizontal lines (branches) in the phylogenetic tree. The further apart viruses are on the horizontal axis of a phylogenetic tree, the more genetically different the viruses are to one another.

Phylogenetic trees of influenza viruses will usually display how similar sequences of the nucleotides for hemagglutinin (HA) genes of the vaccine virus and circulating viruses are to each other.

Figure 1: A phylogenetic tree.

For example, after CDC sequences an influenza A(H3N2) virus collected through surveillance, the virus sequence is cataloged with other sequences that have a similar HA gene (H3) and a similar NA gene (N2). As part of this process, CDC compares the new virus sequence with the other virus sequences and looks for differences among them. CDC then uses a phylogenetic tree to visually represent how genetically similar the A(H3N2) viruses are to each other. In Figure 1, virus b is more genetically similar to virus c than d. Viruses b and c share a common ancestor and the total length of the horizontal branches is short.

CDC performs genetic characterization of influenza viruses year-round. This genetic data is used in conjunction with virus antigenic characterization data and other data, like human serology data, to help determine the vaccine viruses for each year’s flu vaccine. The analysis and selection are made twice each year to recommend vaccine viruses for both the Northern Hemisphere and Southern Hemisphere. In the months leading up to the WHO-facilitated vaccine consultation meetings, where the recommendations are made, CDC collects influenza viruses through surveillance and compares the HA and NA gene sequences of current vaccine viruses against those of circulating flu viruses. This is one of the ways CDC assesses how closely related the circulating influenza viruses are to the viruses the seasonal flu vaccine was formulated to protect against.

Sometimes over the course of a season, circulating influenza viruses will change genetically in such a way that further analyses is needed to determine whether they remain antigenically similar to current vaccine viruses or whether a new virus needs to be included in the next flu season’s vaccine. Many other data, including antigenic characterization findings and human serology data, shape the vaccine selection decisions. Antigenic characterization refers to the analysis of a virus’s reaction with antibodies to help assess how it relates to the vaccine virus and other circulating influenza viruses.

Methods of Flu Genome Sequencing

One influenza sample contains many influenza virus particles that were grown in a test tube and that often have small genetic differences in comparison to one another among the whole population of sibling viruses.

Due to the constantly changing nature of influenza viruses, every sample collected from a patient contains many influenza virus particles that have small genetic differences in comparison to one another. Traditionally, scientists have used a sequencing technique called “the Sanger method” to monitor influenza evolution as part of genetic characterization. Sanger sequencing identifies the predominant genetic sequence among the many influenza viruses found in a virus sample. This means small variations in the population of viruses present in a sample are not reflected in the final result. Newer technologies (such as Next Generation Sequencing, described below) are better suited for detecting small variations in the virus genes and offer advantages for whole genome sequencing.

Since 2014, CDC has been using “Next Generation Sequencing (NGS)” methodologies, which have greatly expanded the amount of information and detail that sequencing analysis can provide.

In a typical year, CDC performs whole genome sequencing on about 7,000 influenza viruses from original clinical samples collected through virologic surveillance. NGS uses advanced molecular detection (AMD) to identify gene sequences from each virus in a sample. Therefore, NGS reveals the genetic variations among many different influenza virus particles in a single sample and can increase the speed and accuracy of sequencing each of the protein coding regions of the virus. This level of detail can directly benefit public health decision-making in important ways, but data must be carefully interpreted by highly trained experts in the context of other available information. See AMD Projects: Improving Influenza Vaccines for more information about how NGS and AMD are revolutionizing flu genome mapping at CDC.

CDC researchers and their colleagues successfully reconstructed the influenza virus that caused the 1918-19 flu pandemic, which killed as many as 50 million people worldwide. A report of their work, “Characterization of the Reconstructed 1918 Spanish Influenza Pandemic Virus,” was published in the October 7, 2005 issue of Science. The work was a collaboration among scientists from CDC, Mount Sinai School of Medicine, the Armed Forces Institute of Pathology, and Southeast Poultry Research Laboratory. The following questions and answers describe this important research and related issues.

Note: For a detailed historical summary of this work, including how it was conducted, the people involved, and the lessons learned from it, see The Deadliest Flu: The Complete Story of the Discovery and Reconstruction of the 1918 Pandemic Virus.

Background on the Research

What research does the Science article describe? Why is it important?

This report describes the successful reconstruction of the influenza A(H1N1) virus responsible for the 1918 “Spanish flu” pandemic and provides new information about the properties that contributed to its exceptional virulence. This information is critical to evaluating the effectiveness of current and future public health interventions, which could be used in the event that a 1918-like virus reemerges. The knowledge from this work may also shed light on the pathogenesis of contemporary human influenza viruses with pandemic potential. The natural emergence of another pandemic virus is considered highly likely by many experts, and therefore insights into pathogenic mechanisms can and are contributing to the development of prophylactic and therapeutic interventions needed to prepare for future pandemic viruses.

What are the reasons for doing these experiments?

The influenza pandemic of 1918-19 killed an estimated 50 million people worldwide, many more than the subsequent pandemics of the 20th century. The biological properties that confer virulence to pandemic influenza viruses have not traditionally been well understood and warranted further study. Research to better understand how the individual genes of the1918 pandemic influenza virus contribute to the disease process provide important insights into the basis of virulence. This kind of information has helped health officials to devise appropriate strategies for early diagnosis, treatment, and prevention, should a similar pandemic virus emerge. Additionally, such research informs the development of general principles with which we can better design antiviral drugs and other interventions against all influenza viruses with enhanced virulence.

Who funded the work described in this article?

Work with the reconstructed 1918 virus was conducted at and supported by CDC. The U.S. Department of Agriculture (USDA), the National Institutes of Health (NIH), and the Armed Forces Institute of Pathology (AFIP) all provided support for many other aspects of this research.

When did CDC begin research on the 1918 virus?

CDC studies of the 1918 influenza virus were begun in 2004 with the initiation of testing of viruses containing subsets of the eight genes of the 1918 virus. Previous articles describing the properties of such viruses were published before 2005. Reconstruction of the entire 1918 virus was begun in August 2005.

Could a 1918-like H1N1 virus re-emerge and cause a pandemic again?

It is impossible to predict with certainty the emergence of a future pandemic, including a 1918-like virus. Pandemics occur when an influenza virus emerges to which there is little, or no, preexisting immunity in the human population. However, it is generally thought that a 1918-like pandemic would be less severe due to the advent of vaccines to prevent flu, current FDA-approved antiviral influenza drugs, and the existing global influenza surveillance system that the World Health Organization maintains.

Are current antivirals and vaccines effective against the 1918 H1N1 virus?

Yes. Oseltamivir (Tamiflu® or generic), has been shown to be effective against similar influenza A(H1N1) viruses and is expected to be effective against the 1918 H1N1 virus. Other antivirals (zanamivir, peramivir and baloxavir) have not been tested against this specific virus but are expected to also be effective. Vaccines containing the 1918 HA or other subtype H1 HA proteins were effective in protecting mice against the 1918 H1N1 virus. Vaccination with current seasonal influenza vaccines is expected to provide some protection in humans since seasonal influenza vaccines provided some level of protection against the 1918 H1N1 virus in mice.

Are new prophylactics and therapeutics that could be effective against the 1918 virus under way?

Scientists continue to work on development of new antivirals which may be effective against a 1918-like virus. The reconstruction of the 1918 H1N1 pandemic virus and subsequent studies that followed showed that the 1918 polymerase genes contribute to efficient replication of the pandemic virus. This insight identified an important virulence factor in the study of influenza that is now targeted for antiviral compound development. Therefore, new polymerase inhibitors promise to add to the clinical management options against influenza virus infections in the future.

Biosafety Precautions

Was the public at risk from the experiments being done on this virus?

The work described in this report was done using stringent biosafety and biosecurity precautions that are designed to protect workers and the public from possible exposure to this virus (for example, from accidental release of the virus into the environment). The 1918 virus used in these experiments has since been destroyed at CDC and does not pose any ongoing risk to the public.

What biosafety and biosecurity precautions for protecting laboratory workers and the public were in place while this work was being done?

Before the experiments were begun, two tiers of internal CDC approval were conducted: an Institutional Biosafety Committee review and an Animal Care and Use Committee review. All viruses containing one or more gene segments from the 1918 influenza virus were generated and handled in accordance with biosafety guidelines of the Interim CDC-NIH Recommendation for Raising the Biosafety Level Laboratory Work Involving Noncontemporary Human Influenza Viruses. Although the 1918 virus was not designated as a select agent at the time this work was performed, all procedures were carried out using the heightened biosecurity elements mandated by CDC’s Select Agent program. The Intra-governmental Select Agents and Toxins Technical Advisory Committee recommended that the reconstructed 1918 influenza virus be added to the list of HHS select agents on September 30, 2005. Following this recommendation, CDC amended its regulations and designated all reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments (reconstructed 1918 Influenza virus) as a select agent.

What are the appropriate biosafety practices and containment conditions for work with the 1918 strain of influenza?

Biosafety Level 3 or Animal Biosafety Level 3 practices, procedures and facilities, plus enhancements that include special procedures (discussed in the next question below), are recommended for work with the 1918 strain. There are four biosafety levels that correspond to the degree of risk posed by the research and involve graded levels of protection for personnel, the environment, and the community. Biosafety Level 4 provides the most stringent containment conditions, Biosafety Level 1 the least stringent. These biosafety levels consist of a combination of laboratory practices and techniques, safety equipment, and laboratory facilities that are appropriate for the operations being performed. The specific criteria for each biosafety level are detailed in the CDC/NIH publication Biosafety in Microbiological and Biomedical Laboratories.

What is Biosafety Level 3 “enhanced”? What are the specific enhancements used for work with the 1918 strain of influenza?

A Biosafety Level 3 facility with specific enhancements includes primary (safety cabinets, isolation chambers, gloves and gowns) and secondary (facility construction, HEPA filtration treatment of exhaust air) barriers to protect laboratory workers and the public from accidental exposure. The specific additional (“enhanced”) procedures used for work with the 1918 strain include:

• Rigorous adherence to additional respiratory protection and clothing change protocols;
• Use of negative pressure, HEPA-filtered respirators or positive air-purifying respirators (PAPRs);
• Use of HEPA filtration for treatment of exhaust air; and
• Amendment of personnel practices to include personal showers prior to exiting the laboratory.

Further details of the biosafety recommendations for work with various human and animal influenza viruses, including 1918 virus, can be found in the interim CDC/NIH guidance for such work at Interim CDC-NIH Recommendation for Raising the Biosafety Level Laboratory Work Involving Noncontemporary Human Influenza Viruses.

How were these experiments conducted safely using containment provided by BSL-3 with enhancements?

Highly trained laboratorians worked with the 1918 influenza virus strain safely using BSL-3-enhanced containment. Researchers at CDC receive specialized training and go through a rigorous biosafety (and security) clearance process. For the work reported in the Science article, the lead CDC researcher provided routine weekly written reports to CDC management officials, including the agency’s Chief Science Officer, and was instructed to notify agency officials immediately of any concerns related to biosafety or biosecurity.

A BSL-3 facility with specific enhancements includes primary (safety cabinets, isolation cabinets, gloves, gowns) and secondary (facility construction) barriers to protect laboratory workers and the public from accidental exposure. Specific enhancements include change-of-clothing and shower-out requirements, and the use of a powered air purifying respirator (PAPR; half body suits). The primary and secondary barriers plus additional personal safety practices provide appropriate containment for conducting such influenza research. CDC evaluated the specific studies to be conducted as well as the highly experienced scientific team conducting the research and concluded that this work could proceed under BSL-3 containment with enhancements.

Why was BSL-3-enhanced containment used for work on the 1918 H1N1 virus when most human influenza viruses of the H1N1 subtype are handled under much less stringent containment?

The appropriate biosafety measures for working a given pathogen depend upon a number of factors, including previous experience with the pathogen or similar pathogens, the virulence and transmissibility of the pathogen, the type of experiment, and the availability of vaccines and/or antimicrobial drugs effective against the pathogen. Prior to reconstruction of the 1918 virus, CDC carefully evaluated the specific studies to be conducted and concluded that this research could safely and securely be done under BSL-3-enhanced containment. All viruses containing one or more gene segments from the 1918 influenza virus were generated and handled under high-containment (BSL 3-enhanced) laboratory conditions in accordance with guidelines of NIH and CDC. The recommendations for biosafety levels are made by a panel of experts and are followed in a stringent manner.

A higher level of containment (biosafety level 4) is utilized for work on novel or exotic pathogens for which there is no treatment or vaccine. This is not the case for the 1918 virus. Descendants of the 1918 influenza virus still circulate today, and current seasonal influenza vaccines provide some protection against the 1918 virus. In addition, two types of antiviral drugs, rimantadine (Flumadine) and oseltamivir (Tamiflu® or generic) have been shown to be effective against similar influenza A(H1N1) viruses and are expected to be effective against the 1918 H1N1 virus. Other antivirals (zanamivir, peramivir and baloxavir) have not been tested against this specific virus but also are expected to be effective.

The physical and engineering design of BSL-3-enhanced containment is very similar to that used in BSL-4 laboratories. The BSL-3 laboratory also has state-of-the-art directional airflow control which filters outgoing air, and all waste is autoclaved or decontaminated before it leaves the work area, preventing escape of infectious agents.

Biosecurity Issues

Did the generation of the 1918 Spanish influenza pandemic virus containing the complete coding sequence of the eight viral gene segments violate the Biological Weapons Convention?

No. Article I of the Biological Weapons Convention (BWC) specifically allows for microbiological research for prophylactic, protective, or other peaceful purposes. Article X of the BWC encourages the “fullest possible exchange of…scientific and technological information” for the use of biological agents for the prevention of disease and other peaceful purposes. Further, Article X of the BWC provides that the BWC should not hamper technological development in the field of peaceful bacteriological activities. Because the emergence of another pandemic virus is considered likely, if not inevitable, characterization of the 1918 virus may enable us to recognize the potential threat posed by new influenza virus strains, and it will shed light on the prophylactic and therapeutic countermeasures that will be needed to control pandemic viruses.

Did the report provide a “blueprint” for bioterrorists to develop and unleash a devastating pandemic on the world?

No. This report does not provide the blueprint for bioterrorist to develop a pandemic influenza strain. The reverse genetics system that was used to generate the 1918 virus is a widely used laboratory technique. While there are concerns that this approach could potentially be misused for purposes of bioterrorism, there are also clear and significant potential benefits of sharing this information with the scientific community: namely, facilitating the development of effective interventions, thereby strengthening public health and national security.

Is the 1918 influenza virus a select agent?

The Intra-governmental Select Agents and Toxins Technical Advisory Committee convened on September 30, 2005, and recommended that the reconstructed 1918 influenza virus be added to the list of HHS select agents. Following this recommendation, CDC amended its regulations and designated all reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments (reconstructed 1918 Influenza virus) as a select agent.

What is the Select Agent Program?

The Centers for Disease Control and Prevention (CDC) regulates the possession, use and transfer of select agents and toxins that have the potential to pose a severe threat to public health and safety. The CDC Select Agent Program oversees these activities and registers all laboratories and other entities in the United States of America that possess, use or transfer a select agent or toxin.

The U.S. Departments of Health and Human Services (HHS) and Agriculture (USDA) published final rules for the possession, use, and transfer of select agents and toxins (42 C.F.R. Part 73, 7 C.F.R. Part 331, and 9 C.F.R. Part 121) in the Federal Register on March 18, 2005. All provisions of these final rules supersede those contained in the interim final rules and became effective on April 18, 2005.