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Influenza A is a single-stranded RNA virus in the Orthomyxoviridae family. This family also includes 2 other types of virus, influenza B and C, which typically produce milder disease, although B is considered important enough that it is included in the annual influenza vaccine formulation. Influenza A virus infects both birds and mammals. Birds are the endemic reservoir for many of the influenza A subtypes. In humans, influenza is a seasonal illness, circulating during the winter months. Importantly, the World Health Organization (WHO) reports that influenza A infects tens of millions of individuals annually, with hundreds of thousands of deaths. In the United States, influenza is responsible for approximately 50,000 deaths annually. The current outbreak involves a new H1N1 type A influenza strain called Novel H1N1 influenza A. This strain has also been called swine flu or swine-origin influenza virus, 2009 H1N1, and Pandemic H1N1.
How Is The Virus Spread?
Influenza viruses are spread from one person to another in 2 main ways. First, as a respiratory tract virus, it is released into the air and onto surfaces by sneezing and coughing. (Figure 1) The particles can be inhaled by another person in close proximity or fall onto objects that are then touched by another person and accidentally introduced into the nose, eyes, and mouth. Also, the infected person can contaminate his or her hands and then spread infection by touching commonly used objects like keyboards and telephones.
Symptoms of Infection
Symptoms of infection typically include abrupt onset of fever, chills, headache, nasal congestion, sore throat, fatigue, and, sometimes, a dry cough. Early on, the symptoms of influenza can resemble those of the common cold, except that fevers are more common and the onset of symptoms is more abrupt with influenza infection. Gastrointestinal symptoms are typically uncommon in adults but are often seen in children with seasonal influenza. Symptoms typically progress to high fever, diffuse myalgias and malaise, which can leave patients bedridden for days. Serious complications include primary viral pneumonia, secondary bacterial pneumonia, myocarditis, and encephalopathy.
Features of the Various Influenza Viruses
The influenza viruses are identified by a number of features. (Figure 2) There is a central core of genetic material, the viral RNA, which is surrounded by a lipid envelope. Embedded in the envelope are 2 main types of glycoproteins. One glycoprotein is hemagglutinin (HA), which mediates binding and entry of the virus into a host cell to be infected. The other is neuraminidase (NA), which allows release of progeny viruses from the infected cell after it undergoes replication within that cell. Below the schematic in Figure 2 is an example of a full virus name. The name begins with the virus type, which is either influenza A, B, or C. In this case, it is type A. This is the type of genetic material contained within the virus. The next part of the name refers to the geographic origin of the virus. Then follows the strain origin, year of isolation, and last, the virus subtype, designated by the type of HA and NA found on its surface.
Figure 3 shows a transmission electron micrograph of influenza viruses, which closely resembles the schematic drawing. The virus is roughly spherical, although filamentous forms can be seen, and the surface is studded with the HA and NA glycoproteins. Based on this image, one can appreciate how these proteins are readily exposed to the immune response, and understand why the majority of the immune response is directed against them.
HA has 16 known types, although only H1, H2, H3, H5, H7, and H9 are known to infect humans at this time. The same virus subtype can have different strains, with very different immunity and pathogenicity profiles. In the 2008 influenza season, 3 influenza viruses were circulating: SH1N1, SH3N2, and NH1N1. The Seasonal H1N1 virus that has been around for more than 100 years is very different from the Novel H1N1 virus that is currently in the news.
Influenza Virus Type, Subtype, and Strain
The type of the virus refers to the composition of the genetic material. Influenza viruses are either A, B, or C. The subtype refers to the HA and NA components of the virus. Seasonal H1N1 and H3N2 influenza A viruses are 2 subtypes of influenza A. The nomenclature gets a bit more complicated when trying to distinguish between the Seasonal H1N1 and Novel H1N1 viruses. Both are the same subtype, but have different geographic origins and genetic composition. They also cause slightly different infections in humans. They are different strains of the same influenza A virus subtype.
Unique Features of Influenza A
Influenza A has a number of unique features that set it apart from other viruses, including its mutability and diversity. Unlike other RNA viruses, within the influenza A virus, 8 distinct RNA segments make up the genome. This is a small genome, encoding for only 11 proteins. The first 3 segments encode for the RNA polymerase, which is responsible for replication of the RNA genome. Next are segments that encode for HA, nucleoprotein, NA, matrix and nonstructural proteins.
Like most RNA viruses, it uses an RNA-dependent RNA polymerase to replicate its RNA. Unlike DNA polymerase, RNA polymerase lacks proofreading ability. So, when mistakes are made during replication, as they are with both RNA and DNA polymerase, there is no method to correct those mistakes. As a result, it is estimated that approximately 1 error in every 10,000 nucleotides occurs in the final replicated RNA product. This is the approximate length of the entire influenza A virus genome. Thus, you can predict that the majority of newly made influenza viruses are mutants.
The numerous mutations of influenza viruses can result in a change in the antigens expressed on the virus’s surface, a phenomenon called antigenic drift. The host’s immune response is directed against these surface antigens. This antigenic diversity is the reason why immunity from previous influenza infection does not necessarily protect against a mutated strain the following year, and why a new vaccination is necessary every year.
In addition to antigenic drift, which can occur with all influenza viruses (A, B, and C), a very different phenomenon, antigenic shift, can occur in influenza A. Unlike antigenic drift, which can introduce minor changes in antigen composition, antigenic shift can introduce a completely new strain of virus. This is due to genetic reassortment, which can occur with some viruses when 2 or more viruses infect a single cell. This is a rare event, but one that can lead to a new virus with a mixed or reassorted genetic composition, and to production of new, drastically different proteins against which there is no preexisting immunity in the general population. This type of genetic mixing can occur in humans or intermediate hosts such as pigs, which have the unique ability to be infected with human, swine, and avian strains of influenza, thus being an ideal mixing vessel for a reassortment event. This can be an important source of new pandemic variants.
Influenza Epidemics and Pandemics
Annual influenza epidemics and global pandemics are the result of antigenic drift and shift. Influenza A causes recurrent epidemics, which are fairly predictable annual infections worldwide. Approximately 3 times per century, influenza infection also causes a pandemic, or an epidemic of larger proportion. Influenza is unique as a pandemic threat, since it can rapidly infect up to 30% of the population in just months. Because of this ability to infect a large number of people in a short time, even a conservative 2% mortality rate could mean 135 million deaths in 1 year. This is 4 times greater than the number of deaths attributed to HIV-1 in the past 30 years!
Spanish Influenza (1918–1919)
The most famous influenza pandemic was the Spanish influenza of 1918–1919. This virus was an H1N1 subtype, like the currently circulating pandemic strain, although quite unique in its antigenic expression and behavior in humans. The Spanish influenza virus is notable in that it caused disease of unprecedented severity, killing up to 50 million people worldwide. In fact, the mortality rates were >2.5%, compared to the typical mortality rate of an annual influenza epidemic, which is <0.1%. In terms of numbers of individuals, consider that 50,000 Americans die each year as a result of annual influenza. The Spanish influenza killed 650,000 Americans. The 1918 strain was thought to be an avian-like virus that entered the population, with subsequent antigenic drift likely causing waves of renewed virulence over the coming years. There are some gaps in our knowledge of this virus, but we know it continued from 1934 to mid-1950, disappeared for awhile, and then reappeared in 1977. A related H1N1 strain is still circulating today as seasonal influenza, distinct from the Novel H1N1 strain. Two to 3 other pandemics have occurred in the last century, depending on one’s definition of a pandemic. All had considerably lower mortality than the 1918 influenza.
Asian Influenza (1957)
The Asian influenza of 1957 was an H2N2 subtype of influenza A virus. It was a human/avian reassortant, and caused 1.5 million fatalities worldwide. Interestingly, H2 is no longer circulating in the population.
Hong Kong Influenza (1968–1970)
Next was the so-called Hong Kong influenza from 1968 to 1970. This was an H3N2 subtype, and also a human/avian reassortant. Of note, this strain never left circulation, and the current Seasonal H3N2 is a variant of this virus.
Swine Flu (1976)
The influenza that occurred in 1976 was referred to as a swine flu. The emergence of that strain caused considerable public apprehension as a result of media coverage and sparked a massive vaccination campaign, but never amounted to much of anything.
Russian Influenza (1977)
Then came the milder Russian influenza of 1977. Because of the lower mortality and the fact that it only infected children, this was not considered a true pandemic.
Novel H1N1 Influenza A Outbreak (2009)
The current pandemic, caused by a new strain of H1N1, is called the Novel H1N1 virus. It has also been called the swine flu or swine-origin influenza virus, since it is thought to have originated in swine before mutating to be transmissible between humans. Other names for the virus include 2009 H1N1 and Pandemic H1N1.
The likely origin of this outbreak was La Gloria, Veracruz, Mexico, in February 2009. However, it wasn’t until early April 2009 that Mexican health authorities began investigating a high numbers of cases of respiratory illness, which affected about 28% of the population in La Gloria. At that time, the Pan American Health Organization, a regional office of the WHO, was notified and Mexican health authorities closed down many private and public businesses in Mexico City in an attempt to contain the spread of the virus. Unfortunately, it was too late because the virus had already spread outside of Mexico.
On April 21, the Centers for Disease Control and Prevention (CDC) reported the first US cases in 2 children, and by May 11, the virus had spread to 30 countries. Just 10 days later, Novel H1N1 had spread to 41 countries, and on June 11, the WHO declared a pandemic alert phase 6, the highest alert on the pandemic scale. An important consideration is that the pandemic alert indicates geographic spread, and not severity of illness. In this case, the WHO noted that new cases were occurring in people without a travel history to Mexico or contact with travelers, and the organization concluded that continued spread was inevitable.
Influenza A is primarily a winter virus, and all of the influenza cases from previous seasons (2006–2008) peaked between weeks 5 to 7 of the new year. What was different about the most recent season was that there was a second peak, long after the typical influenza case peak. Almost all of the cases at that time were the Novel H1N1 strain. The CDC estimates that 1 million people became ill with Novel H1N1 flu between April and June 2009 in the United States, based on the cases that were reported to local government health agencies.
Swine Flu and Novel H1N1 Influenza A
A phylogenetic analysis published by Smith et al in Nature, June 2009, showed that Novel H1N1 was derived from several swine viruses that were circulating for more than 10 years—hence, the original nomenclature, swine flu. However, some of the segments came from a swine virus that had been circulating in North America, which was already a triple reassortment of swine, avian, and human influenza viruses. The other segments came from a Eurasian swine virus with avian components. So, in fact, the Novel H1N1 virus is a complex mixture of human, swine, and avian components. Therefore, the term swine flu is not exactly accurate. Furthermore, no evidence indicates that the current virus is endemic to swine or is transmitted from swine to humans. Novel H1N1 virus truly is a new reassortant that is transmitted primarily from human to human, and not from swine to human. Also, exposure to swine is not a risk factor. Unfortunately, considerable confusion occurred early on regarding the relationship of the virus to swine and pork products, and many animals were unnecessarily killed in some countries in a misguided attempt to slow the spread of the virus.
What is the Difference Between Seasonal H1N1 and Novel H1N1 Influenza A?
The genetic reassortment leading to Novel H1N1 virus has produced what has been termed a “pseudoshift” instead of true antigenic shift. It is not a new HA type, so technically this does not represent antigenic shift. Yet it is significantly different from the Seasonal H1N1. The divergence between Seasonal and Novel H1 is <25%, more than the typical variation seen among human H1 strains, but much less than what is seen between different HA types. For example, the typical variation between H1 and H3 is 40% to 60%. Technically, this new virus is antigenically distinct from seasonal viruses, but is not distinct enough to have a new HA type. Therefore, we are left with the confusing situation in which we have a Novel H1N1 strain and a Seasonal H1N1 strain—both of which will be circulating this coming winter. They share the same subtype, but are different strains and behave somewhat differently.
As the pandemic has progressed, evidence indicates that the epidemiology is very different from what is seen with seasonal influenza and, symptomatically, the outbreak has differed in the higher prevalence of gastrointestinal symptoms such as vomiting and diarrhea. In particular, children and young adults appear to be at a disproportionate risk of infection and severe disease, while older people have been less affected than is typically seen in seasonal influenza. There is evidence that some individuals >60 years old have preexisting immunity to the Novel H1N1 flu virus, perhaps from exposure to previous swine flu. With the current outbreak, the highest infection rates are in children and young adults <25 years old, whereas the >65 age group has remarkably few infections. Between April 15 and July 24 of 2009, the highest number of hospitalizations was among children aged 0 to 4 years, followed by the 5 to 24 age group. The hospitalization rate for people in the 25 to 49 age group was lowest at 1.1 per 100,000 people. These numbers are lower than what is typically seen during a normal influenza season, indicating that infection, in general, is rather mild compared to seasonal influenza. Also important to note is that those who were hospitalized were significantly more likely to have underlying medical conditions such as asthma, diabetes, or heart disease. Interestingly, obesity has also been identified as a risk factor, whereas it has not traditionally been a risk factor for severe influenza disease. Pregnancy also increases the risk of severe disease and hospitalization.
The CDC examined the hospital records of 268 patients hospitalized with Novel H1N1 influenza early during the outbreak and calculated the number of deaths from this group. Deaths were highest among people aged 25 to 49. This pattern is very different from what is typically seen in seasonal influenza, where an estimated 90% of influenza-related deaths occur in people 65 years of age and older. So it seems that the Novel H1N1 influenza A infects a disproportionate number of young adults and children compared to seasonal influenza, but kills more individuals in the 25 to 49 year age range, and that individuals over the age of 64 do not seem to be at increased risk of severe disease as they normally are with seasonal influenza.
Where Do We Stand With Novel H1N1 Virus?
The Novel H1N1 strain is now the dominant strain worldwide. It has passed into the southern hemisphere, and has peaked in several countries including Chile, Argentina, New Zealand, and Australia. Still other countries, such as South Africa and Bolivia, continue to have a high number of cases with no signs of decline. Central America and tropical Asia also continue to see sustained high levels of disease.
In Canada and the United States, influenza activity with Novel H1N1 is picking up again, and many areas are reporting widespread disease. Europe also has areas of widespread activity, and Japan has passed its seasonal epidemic threshold already, signaling an early start to its annual influenza season. The expectation is that the Novel H1N1 strain is here to stay. The current situation is rapidly evolving and the CDC (http://www.cdc.gov/h1n1flu/) or WHO (http://www.who.int/csr/disease/swineflu/updates/en/) Web sites contain the most up-to-date information.
Diagnosis and Testing
Definitive diagnosis of influenza cannot be done by clinical symptoms alone, but requires proven laboratory techniques such as viral tube culture, which is still the gold standard for diagnosis. Unfortunately, several days are necessary to get a result, which is long after treatment should be initiated. There also are shell vial culture assays, which decrease the detection time to 1 to 2 days, but this time frame is still fairly long compared with some of the more rapid tests that are available. Some laboratories are using direct immunofluorescence method for detection of virus A antigen in clinical respiratory samples, and, with strict quality control measures, have reported very high sensitivity and specificity for detection of influenza A virus. However, the technique is time consuming, labor intensive, takes highly trained personnel to perform and interpret the test, and is not ideally suited for large-volume laboratories. In addition, neither culture nor immunofluorescent tests provide subtyping and drug susceptibility information.
Rapid Influenza Diagnostic Tests
Rapid influenza diagnostic tests (RIDTs) deserve a special mention because they are so widely used throughout the United States. These tests detect viral nucleoprotein antigen directly from the patient’s respiratory sample, and provide results in 30 minutes or less, which is ideal in the patient care setting. Unfortunately, they have 2 main drawbacks. First, like culture, they do not provide subtyping or antiviral susceptibility information. Second and more importantly, they have very low sensitivity rates, ranging anywhere from 10% to 70%, depending on the test and particular study. There may be a greater sensitivity in children since this population tends to shed a larger amount of virus. Regardless, a negative result does not rule-out infection, and the CDC specifically states that:
“If clinical suspicion of influenza is high in a patient who tests negative by RIDT, empiric antiviral therapy should be administered, if appropriate, and infection control measures implemented.”
This statement calls into question the utility of RIDTs. In contrast to sensitivity, specificity is generally high, although false-positive results can occur, especially during periods of low activity, where there is a low likelihood of influenza disease.
Because of these drawbacks, confirmation of negative results, and in some cases, positive results, is recommended for the RIDTs. This is usually done by culture or PCR. Much discussion has focused recently on whether RIDTs should be used at all, given their significant limitations. However, many have argued that, in the right clinical setting, a positive result provides useful information and allows for immediate treatment and the implementation of infection control measures. Pediatricians are probably the biggest proponents of RIDTs, since these tests may work better in the pediatric population. It is important to note that the RIDTs are capable of detecting the Novel H1N1 influenza A virus, although the sensitivity of detection may be slightly less than what is reported for seasonal influenza A viruses.
Finally, there are molecular tests—in this case, reverse transcription PCR (RT-PCR).
In RT-PCR, viral RNA in a sample is first converted to cDNA, and then amplified so that it can be readily detected. Many tests are capable of detecting the Novel H1N1 strain. However, the first assay to actually distinguish Novel H1N1 specifically from the other seasonal influenza strains was produced by the CDC.
On April 26, 2009, the Secretary of the Department of Heath and Human Services determined that a public health emergency existed and, the following day, the US Food and Drug Administration (FDA) granted emergency-use authorization for the CDC RT-PCR Influenza A panel. This was available initially only to “qualified” health laboratories (government-based laboratories) that had undergone training at the CDC.
However, the assay specifics are now publicly available and can be purchased and validated by private health laboratories that choose to do so. Since this time, the FDA has decided to grant emergency-use authorization for other real-time PCR tests. Some other formats are more user friendly for commercial labs, and a number of companies are seeking, or have already received, this authorization. So we can expect to see a variety of influenza PCR assays become available for general purchase.
Currently, it is probable that any positive influenza result by our PCR assay #88544 Influenza Virus Type A and Type B RNA by Rapid PCR represents the Novel strain, since greater than 99% of all infections in the United States are caused by this strain.
One may ask why it is even important to differentiate the influenza virus subtypes. The answer lies in the varying antiviral susceptibility patterns seen with the different viruses and the need for differing treatments.
The decision to treat infected individuals is at the discretion of the care provider, and WHO states that antiviral treatment is not necessary for healthy adults and children with uncomplicated disease. Therefore, treating everyone is not necessary. This could become quite important if the 2009–2010 influenza season becomes widespread, with a larger percentage of the population becoming infected. In this scenario, antiviral drugs could be in short supply, and treatment would have to be limited to those who are very ill.
Currently, 2 main classes of antiviral drugs are active against influenza A virus: the ion channel inhibitors such as amantadine and rimantadine, and the neuraminidase inhibitors oseltamivir and zanamivir. Novel H1N1 is widely susceptible to the neuraminidase inhibitors, and both are recommended for treatment of patients at risk for severe disease. Oseltamivir is perhaps the most widely used drug and is known best under the trade name Tamiflu. Zanamivir, marketed under the trade name Relenza, is also an effective drug, but is administered through an inhaler and cannot be given to certain patient groups, such as those with preexisting respiratory conditions. The ion-channel inhibitors or adamantanes are not a good choice for Novel H1N1 treatment, since this strain is uniformly resistant to them.
In contrast to the widespread susceptibility of Novel H1N1 to oseltamivir, Seasonal H1N1 is widely resistant to this drug. This is a rather new phenomenon, since there was only a 0.7% resistance rate in the 2006–2007 influenza season, but a 98.5% resistance rate in the 2008–2009 influenza season. Fortunately, Seasonal H1N1 is still susceptible to the adamantanes, such as rimantadine, and the inhaled neuraminidase inhibitor zanamivir. So, there are still therapeutic options for these patients.
Finally, Seasonal H3N2 is widely susceptible to oseltamivir, but mainly resistant to the adamantanes. This variation in susceptibility patterns provides increasing justification for diagnostic tests that can provide subtyping information, so that the appropriate therapy can be chosen. The alternative is to treat all patients with combination therapy, with the idea that at least 1 of the drugs will be active against the infection. This isn’t a bad approach, but may increase the likelihood that patients experience negative side effects.
A new twist in the story is that a small number of Novel H1N1 cases with resistance to oseltamivir have been detected worldwide cases in the United States. Most of these patients had been exposed to oseltamivir treatment and had presumably developed resistance during the course of treatment. Fortunately, there is no evidence of onward transmission of the resistant strains to other people. Pyrosequencing of these few resistant isolates has shown the NA gene to contain an H274Y amino acid substitution, which has been associated with oseltamivir resistance in Seasonal H1N1. As expected, the WHO and CDC are closely monitoring the situation to determine if more resistant cases arise.
Vaccines and Prevention
As always, prevention is preferred to treatment, and vaccines remain the best method for preventing outbreaks of influenza. Vaccines are currently given annually to humans in developed countries and to farmed poultry in some countries. The seasonal vaccines include the most prevalent strain of human influenza A subtypes (Seasonal H1N1 and Seasonal H3N2) and influenza B, making it a trivalent vaccine. Because of antigenic drift, the vaccine is revised every 1 to 3 years to reflect what is predicted to match the circulating strain that year. Usually the prediction is fairly accurate, but it is not uncommon to see some mismatch between one of the vaccine strains and the actual circulating strain.
The inactivated virus vaccine has been used for decades, with varying efficacy. It has a 60% to 80% efficacy in children and young adults, but a decreased efficacy in adults >60 years, which, unfortunately, are the individuals who usually need it the most. There is also now a live attenuated vaccine for individuals aged 2 to 49, which may offer enhanced immunity. Further studies are needed on this subject.
The emergence of Novel H1N1 took the world by surprise, and so it was too late to incorporate the Novel H1N1 strain into vaccine preparations for fall 2009. Instead, there will be a second vaccine that specifically protects against the Novel H1N1 strain, which should be available by mid to late October 2009. The CDC recently published recommendations for the use of this new vaccine, including the individuals who should receive it, based on their risk for severe disease:
According to this document, the 5 initial groups that will be targeted for vaccination are:
Preventive measures are recommended for all age groups. Frequent hand washing is probably the most important measure, since virus is spread when hands come in contact with infected surfaces and then inoculate mucosal membranes such as the eyes, nose, and mouth. Washing with soap and water, or using a liquid alcohol-based rub is very effective when done for at least 15 seconds. Symptomatic individuals should also be encouraged to wash their hands frequently to prevent spreading virus to surfaces, and to cover their coughs and sneezes, since virus particles spread through large respiratory droplets. There is also a role for surface disinfection by sunlight, disinfectants, and detergents, to help prevent viral spread.
In the clinical setting, the CDC recommends that health care workers wear appropriate masks (eg, N95s) whenever they may potentially be exposed to sick patient generating aerosols. It is still unknown whether the use of other masks, such as surgical masks that tie behind the head, provide any protection from infected individuals, but they may be useful when given to infected patients to wear to contain their respiratory secretions. The CDC publishes updated guidelines regularly on infection control practices, so please reference their Web site for the most recent recommendations.
Despite the wealth of information that is currently available on this new virus, some important questions have not been answered. For example, will subsequent mutations result in increased virulence over time? This was seen with the Spanish influenza of 1918, where subsequent mutations resulted in recurring waves of infection throughout the population. Will double infection with Novel H1N1 and Seasonal H1N1 or Seasonal H3N2 occur? Until this year, Seasonal H1N1 and H3N2 were cocirculating during the typical influenza season, and there is no reason to believe that they won’t return this coming winter. This would mean that we would potentially have 3 influenza viruses, all circulating at the same time. At this point, the risk for reassortment events among these 3 viruses is unknown, but it is possible whenever more than 1 virus is infecting the same host. Alternately, the Novel H1N1 might dominate the scene completely, and the other viruses will not be encountered. Another unanswered question is whether the Novel H1N1 will begin to circulate in a seasonal pattern as some degree of immunity in the population develops, or if it will remain present to some extent all year. Finally, we don’t know if resistance to oseltamivir will spread among the Novel H1N1 virus. This is, of course, one of the biggest fears, since we have a limited number of antiviral drugs that are effective against influenza A viruses. Only time will bring the answer to these questions. Until then, we need to continue improving the diagnostic and therapeutic options, while maintaining ongoing surveillance of influenza activity around the world.
This information is based on a Mayo Medical Laboratories’ Hot Topic, recorded on September 15, 2009. The information has been updated to reflect the status of the novel H1N1 pandemic prior to printing (Oct 2009).
Authored by Dr. Bobbi Pritt
Images provided by the CDC.
Figure 1: James Gathamy
Figure 3: C.S. Goldsmith and A. Balish