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19.3B: Influenza - Biology

19.3B: Influenza - Biology



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Influenza is a viral infection of the lungs characterized by fever, cough, and severe muscle aches. Influenza is not a case of low fever and sniffles that keeps you home in bed for a day nor a gastrointestinal upset ("stomach flu").

Influenza was responsible for the most devastating plague in human history - the "Spanish" flu that swept around the world in 1918 killing 675,000 people in the U.S. and an estimated 20–50 million people worldwide. (A disease that attacks a large fraction of the population in every region of the world is called a pandemic.) (It is uncertain where the flu first appeared, but it certainly wasn't in Spain.)

No one at the time even knew what disease agent was causing the pandemic. Not until 1930 (in pigs) and 1933 (in humans) was it established that influenza is caused by a virus.

This electron micrograph (courtesy of Dr. K. G. Murti) shows several influenza virus particles (at a magnification of about 265,000x). The surface projections are molecules of hemagglutinin and neuraminidase (see below).

There are three types of influenza:

  • Common but seldom causes disease symptoms.
  • Often causes sporadic outbreaks of illness, especially in residential communities like nursing homes.
  • Responsible for regular outbreaks, including the one of 1918. Influenza A viruses also infect domestic animals (pigs, horses, chickens, ducks) and some wild birds.

The Influenza A Virus

The influenza A virion is

  • a globular particle (about 100 nm in diameter)
  • sheathed in a lipid bilayer (derived from the plasma membrane of its host)
  • Studded in the lipid bilayer are three integral membrane proteins
    • some 500 molecules of hemagglutinin ("H")
    • some 100 molecules of neuraminidase ("N")
    • the M2 membrane protein (not shown).
  • Encased by the lipid bilayer are
    • some 3000 molecules of matrix protein
    • 8 pieces or segments of RNA

Each of the 8 RNA molecules is associated with

  • many copies of a nucleoprotein
  • the three subunits of its RNA polymerase
  • some "non-structural" protein molecules of uncertain function

The Genes of Influenza A

The 8 RNA molecules (the number in brackets is the designated segment number):

  • The HA gene [4] encodes the hemagglutinin. 3 distinct hemagglutinins (H1, H2, and H3) are found in human infections; 15 others have been found in animal flu viruses.
  • The NA gene [6] encodes the neuraminidase. 2 different neuraminidases (N1 and N2) have been found in human viruses; 9 others in other animals.
  • The NP gene [5] encodes the nucleoprotein. Influenza A, B, and C viruses have different nucleoproteins.
  • The M gene [7] encodes two proteins (using different reading frames of the RNA): a matrix protein (M1 — shown in blue) and an ion channel (M2) spanning the lipid bilayer (not shown).
  • The NS gene [8] encodes two different non-structural proteins (also by using different reading frames). These are found in the cytosol of the infected cell but not within the virion itself.
  • One RNA molecule (PA [3], PB1 [2], PB2 [1]) encoding each of the 3 subunits of the RNA polymerase. Occasional frameshifts during translation of PA produce a protein that reduces host gene expression.

The Disease

The influenza virus invades cells of the respiratory passages.

  • Its hemagglutinin molecules bind to sialic acid residues on the glycoproteins exposed at the surface of the epithelial cells of the host respiratory system.
  • The virus is engulfed by receptor mediated endocytosis.
  • The drop in pH in the endosome (endocytic vesicle) produces a change in the structure of the viral hemagglutinin enabling it to
  • fuse the viral membrane with the vesicle membrane.
  • This exposes the contents of the virus to the cytosol.
  • The RNA enter the nucleus of the cell where fresh copies are made.
  • These return to the cytosol where some serve as messenger RNA (mRNA) molecules to be translated into the proteins of fresh virus particles.
  • Fresh virus buds off from the plasma membrane of the cell (aided by the M2 protein) thus
  • spreading the infection to new cells.

The result is a viral pneumonia. It usually does not kill the patient (the 1918 pandemic was an exception; some victims died within hours) but does expose the lungs to infection by various bacterial invaders that can be lethal. Before the discovery of the flu virus, the bacterium Hemophilus influenzae was so often associated with the disease that it gave it its name.

Pandemics and Antigenic Shift

Three pandemics of influenza have swept the world since the "Spanish" flu of 1918.

  • The "Asian" flu pandemic of 1957;
  • the "Hong Kong" flu pandemic of 1968;
  • the "Swine" flu pandemic that began in April of 2009.

The pandemic of 1957 probably made more people sick than the one of 1918. But the availability of antibiotics to treat the secondary infections that are the usual cause of death resulted in a much lower death rate. The hemagglutinin of the 1918 flu virus was H1, its neuraminidase was N1, so it is designated as an H1N1 "subtype". Here are some others.

Some strains of influenza A
DateStrainSubtypeNotes
1918H1N1pandemic of "Spanish" flu
1957A/Singapore/57H2N2pandemic of "Asian" flu
1962A/Japan/62H2N2epidemic
1964A/Taiwan/64H2N2epidemic
1968A/Aichi/68H3N2pandemic of "Hong Kong" flu
1976A/New Jersey/76H1N1swine flu in recruits
1977A/USSR/77H1N1"Russian" flu
2009A/California/09H1N1pandemic of "swine" flu [now designated A(H1N1)pdm09]

Until 2009, these data suggest that flu pandemics occur when the virus acquires a new hemagglutinin and/or neuraminidase. For this reason, when an H1N1 virus appeared in a few recruits at Fort Dix in New Jersey in 1976, it triggered a massive immunization program (which turned out not to be needed). However, an H1N1 virus appeared the following year (perhaps escaped from a laboratory) causing the "Russian" flu. We now know that this virus was a direct descendant of the 1918 flu. While accumulating mutations that made it less dangerous, it had been infecting humans until it was replaced by the H2N2 "Asian" flu of 1957. Because most people born before the Asian flu pandemic of 1957 had been exposed to the H1N1 viruses circulating before, the Russian flu primarily affected children and young adults. For the same reason, this pattern was also seen in the 2009-10 pandemic of "swine" flu.

Where do the new H or N molecules come from?

Birds appear to be the source. Both the H2 that appeared in 1957 and the H3 that appeared in 1968 came from influenza viruses circulating in birds. The encoding of H and N by separate RNA molecules probably facilitates the reassortment of these genes in animals simultaneously infected by two different subtypes. For example, H3N1 virus has been recovered from pigs simultaneously infected with swine flu virus (H1N1) and the Hong King virus (H3N2). Probably reassortment can also occur in humans with dual infections.

Epidemics and Antigenic Drift

No antigenic shifts occurred between 1957 ("Asian") and 1968 ("Hong Kong"). So what accounts for the epidemics of 1962 and 1964? Missense mutations in the hemagglutinin (H) gene. Flu infections create a strong antibody response. After a pandemic or major epidemic, most people will be immune to the virus strain that caused it. The flu virus has two options:

  • wait until a new crop of susceptible young people comes along
  • change the epitopes on the hemagglutinin molecule (and, to a lesser degree, the neuraminidase) so that they are no longer recognized by the antibodies circulating in the bodies of previous victims.
    • By 1972, the H3 molecules of the circulating strains differed in 18 amino acids from the original "Hong Kong" strain
    • By 1975, the difference had increased to 29 amino acids.

The gradual accumulation of new epitopes on the H (and N) molecules of flu viruses is called antigenic drift. Spontaneous mutations in the H (or N) gene give their owners a selective advantage as the host population becomes increasingly immune to the earlier strains.

Flu Vaccines

Although a case of the flu elicits a strong immune response against the strain that caused it, the speed with which new strains arise by antigenic drift soon leaves one susceptible to a new infection. Immunization with flu vaccines has proved moderately helpful in reducing the size and severity of new epidemics.

Some vaccines incorporate inactivated virus particles; others use the purified hemagglutinin and neuraminidase. Both types incorporate antigens from the three major strains in circulation, currently:

  • an A strain of the H1N1 subtype
  • an A strain of the H3N2 subtype and
  • a B strain.

Because of antigenic drift, the strains used must be changed periodically as new strains emerge that are no longer controlled by people's residual immunity.

The process:

  • Chicken eggs are infected with the virus expressing the new H and/or N and simultaneously infected with a stock flu virus that grows very well in eggs.
  • Genetic reassortment produces some viruses with both the new H and N genes along with the 8 other genes from the stock strain.
  • This new virus is then grown in massive amounts and the H and N proteins purified for the new vaccine.

The whole process takes several weeks. A promising way to speed things up is to chemically synthesize the new H and N genes and substitute them for the H and N genes in the stock virus. The new virus can be ready for vaccine production in a few days.

Strains used in vaccines for the flu seasons shown.
SeasonH1N1H3N2Type B
86–87A/Chile/83*A/Mississippi/85B/Ann Arbor/86
* As the 86–87 season got underway, it was found that A/Chile/83 no longer gave protection so A/Taiwan/86 was offered as a second shot late in that season.
87–88A/Taiwan/86A/Leningrad/86B/Ann Arbor/86
88–89A/Taiwan/86A/Sichuan/87B/Victoria/87
89–90A/Taiwan/86A/Shanghai/87B/Yamagata/88
90–91A/Taiwan/86A/Shanghai/89B/Yamagata/88
91–92A/Taiwan/86A/Beijing/89B/Panama/90
92–93A/Texas/91A/Beijing/89B/Panama/90
93–94unchangedunchangedunchanged
94–95A/Texas/91A/Shandong/93B/Panama/90
95–96A/Texas/91A/Johannesburg/94B/Harbin/94
96–97A/Texas/91A/Nanchang/95B/Harbin/94
97–98A/Johannesburg/96A/Nanchang/95B/Harbin/94
98–99A/Beijing/95A/Sydney/97B/Beijing/93
99–00A/Beijing/95A/Sydney/97B/Yamanashi/98
00–01A/New Caledonia/99A/Panama/99B/Yamanashi/98
01–02A/New Caledonia/99A/Panama/99B/Victoria/00 or similar
02–03A/New Caledonia/99A/Moscow/99B/Hong Kong/2001
03–04A/New Caledonia/99A/Moscow/99B/Hong Kong/2001
04–05A/New Caledonia/99A/Fujian/2002B/Shanghai/2002
05–06A/New Caledonia/99A/California/2004B/Shanghai/2002
06–07A/New Caledonia/99A/Wisconsin/2005B/Malaysia/2004
07–08A/Solomon Islands/06A/Wisconsin/2005B/Malaysia/2004
The B/Malasia component of the vaccine provided no protection at all. So all three components of the 08–09 vaccine were changed as shown on the next line.
08–09A/Brisbane/2007A/Brisbane/2007B/Florida/2006
09–10A/Brisbane/2007A/Brisbane/2007B/Brisbane/2008
Because the 2009–2010 pandemic of the newly-emerged "swine flu" virus drove the "seasonal" H1N1 viruses (e.g., A/Brisbane/2007) to near extinction,
the "swine flu" H1N1 – now called A(H1N1)pdm09 – replaced the "seasonal" H1N1 in the 10–11 vaccine.
10–11A/California/2009A/Perth/2009B/Brisbane/2008
11–12All three components were unchanged from the previous year
12–13A/California/2009A/Victoria/2011B/Wisconsin/2010
13–14A/California/2009A/Victoria/2011B/Massachusetts/2012
14–15A/California/2009A/Texas/2012B/Massachusetts/2012
15–16A/California/2009A/Switzerland/2013B/Phuket/2013
16–17A/California/2009A/HongKong/2014B/Brisbane/2008
Several vaccines for the 2016-17 season will be quadrivalent; that is, contain a fourth component B/Phuket/2013.

FluMist®

On 17 June 2003, the U. S. Food and Drug Administration (FDA) approved FluMist® – a live-virus vaccine that is given as a spray up the nose. The viruses have been weakened so that they do not cause illness, but are able to replicate in the relatively cool tissues of the nasopharynx where they can induce an immune response. Presumably this is tilted towards IgA production, a better defense against infection by inhaled viruses than blood-borne IgG antibodies. In any case, FluMist® induces a more rapid response than inactivated vaccine and there is some evidence that it provides better protection against antigenic drift as well.

All three currently-circulating strains of flu (H1N1, H3N2, and B) are included. As new strains appear, they can be substituted.

At present, this new vaccine (technically known as LAIV "Live Attenuated Influenza Vaccine") is only approved for children older than 24 months and adults younger than 50. People with immunodeficiency (e.g., AIDS) should also be cautious about taking it.

Update: For as yet unknown reasons, the nasal spray did not work during the 2015–2016 season, and it is not recommended for the upcoming season.

Flublok®

On 16 January 2013, the U. FDA approved an entirely new type of vaccine. Flublok® is made in cell cultures transformed with recombinant DNA encoding the hemagglutinins of the 3 currently circulating flu strains (H1N1, H3N2, and B). The final concentration of antigens is three times that in the current vaccine. Cultures of insect cells are used so there is no problem with possible egg allergies in those receiving the vaccine.

Other weapons against flu

It takes a while for the flu vaccine to build up a protective level of antibodies. What if you neglected to get your flu shot and now an epidemic has arrived?

Amantadine and Rimantadine

These drugs inhibit the M2 matrix protein needed to get viral RNA into the cytosol. They work against A strains only, and resistance to the drugs evolves quickly. By the 2009-2010 flu season, virtually all strains of both H3N2 and H1N1 had developed resistance.

Zanamivir (Relenza®) and Oseltamivir (Tamiflu®)

These drugs block the neuraminidase and thus inhibit the release and spread of fresh virions. Spraying zanamivir into the nose or inhaling it shortens the duration of disease symptoms by one to three days. Unfortunately, by the 2008-2009 flu season, all H1N1 strains circulating in the U.S. had become resistant to Tamiflu.

Antibiotics

Antibiotics are of absolutely no value against the flu virus. However, they are often given to patients to combat the secondary bacterial infections that occur and that are usually the main cause of serious illness and death.

Why so few drugs?

The mechanisms by which amantadine and zanamivir work provide a clue. There are far fewer anti-viral drugs than antibacterial drugs because so much of the virus life cycle is dependent on the machinery of its host. There are many agents that could kill off the virus, but they would kill off host cell as well. So the goal is to find drugs that target molecular machinery unique to the virus. The more we learn about these molecular details, the better the chance for developing a successful new drug.

The "Spanish" Flu

Jeffery Taubenberger and his colleagues have sequenced the genes of the influenza virus that had been recovered from

  • preserved lung tissue of a U.S. soldier who died from influenza in 1918
  • lung tissue from a flu victim whose body had remained frozen in the permafrost of Alaska since she died in 1918

But even with all of its genes now completely sequenced, why the 1918 strain was so deadly is not fully understood. But deadly it is. They have even been able to replace the 8 genes of a laboratory strain of flu virus with all 8 genes of the 1918 strain (using strict biosafety containment procedures!). The resulting virus kills mice faster than any other human flu virus tested. (Reported in the 7 October 2005 issue of Science.)

The Swine Flu of 2009

A new H1N1 flu began infecting humans in North America in April 2009 and has now spread throughout much of the world. Sequencing its genome revealed a novel virus - now called A(H1N1)pdm09 - that contained genes previously found in four different strains of swine flu:

  • an HA gene (H1) derived from the swine flu of 1930 (and closely-related to the H1 of the great 1918 "Spanish" flu pandemic) along with an NP and NS gene from that virus;
  • an NA gene (N1) from a virus that had been circulating in the pigs of Europe and Asia since 1979 along with the M gene from that virus;
  • a PA and PB2 gene that entered pigs from birds around 1998;
  • a PB1 gene that passed from birds to humans around 1968 and from us to pigs around 1998.

Why this remarkable assortment of genes has enabled he virus to jump so successfully from pigs to humans remains to be determined.

The amino acid sequence around the critical epitopes of its H1 molecules closely resemble those found in the resurrected 1918 flu virus. This would explain why

  • Antibodies from elderly survivors of the 1918 pandemic neutralize the new swine flu virus.
  • Antibodies (raised in mice) to the new swine flu virus neutralize the resurrected 1918 flu virus.
  • The recent pandemic caused serious illness and death mostly in young adults and least in children and the elderly. As for the elderly, this contrast to the usual pattern arose because people over 65, even if not old enough to have been exposed to the 1918 virus, had been exposed to H1 viruses that until 1957 had only drifted from the original 1918 virus, and thus they had developed partial immunity. The antibodies in young adults were specific for seasonal flu strains circulating since 1957. These were unable to protect them against the 2009 virus but may have formed damaging immune complexes with them. Youngsters had no anti-flu antibodies and did not form such immune complexes.

"Bird Flu"

Many influenza A viruses are found in birds, both domestic and wild. Most of these cause little or no illness in these hosts. However, some of their genes can enter viruses able to infect domestic animals, as was the case for the PA and PB2 genes of the swine flu of 2009 (above).

On several occasions, bird flu viruses have also infected humans, often with alarmingly-high fatality rates. In 2003, human cases of an H7N7 bird flu virus infection occurred in the Netherlands, and in the same year an H5N1 bird virus caused human cases in large areas of Asia. Most of the human cases seemed to have been acquired from contact with infected birds rather than from human-to-human transmission.

And now in 2013, a new bird flu virus, H7N9, has appeared in humans in China. By the end of the summer of 2013, it had caused 135 observed cases (no one knows yet whether there may also be infected people who are not sick enough to show up at hospitals). 45 of the observed cases were fatal. The victims appear to have been infected through contact with infected poultry with little or no evidence of human-to-human transmission.

As a glance at the tables above will show, humans have had long experience with infections and vaccines by both H1 and H3 flu viruses. But the human population has absolutely no immunity against any H7 viruses. If this virus develops the capability to spread efficiently from human to human, it could lead to another worldwide pandemic.


Systems biology of vaccination for seasonal influenza in humans

Here we have used a systems biology approach to study innate and adaptive responses to vaccination against influenza in humans during three consecutive influenza seasons. We studied healthy adults vaccinated with trivalent inactivated influenza vaccine (TIV) or live attenuated influenza vaccine (LAIV). TIV induced higher antibody titers and more plasmablasts than LAIV did. In subjects vaccinated with TIV, early molecular signatures correlated with and could be used to accurately predict later antibody titers in two independent trials. Notably, expression of the kinase CaMKIV at day 3 was inversely correlated with later antibody titers. Vaccination of CaMKIV-deficient mice with TIV induced enhanced antigen-specific antibody titers, which demonstrated an unappreciated role for CaMKIV in the regulation of antibody responses. Thus, systems approaches can be used to predict immunogenicity and provide new mechanistic insights about vaccines.

Annual vaccination is one of the most effective methods for preventing influenza 1 . At present, two types of vaccines for seasonal influenza are licensed for use in the USA: trivalent inactivated influenza vaccine (TIV), given by intramuscular injection and live attenuated influenza vaccine (LAIV), administered intranasally. These vaccines contain three strains of influenza viruses that are usually changed annually on the basis of the results of global influenza surveillance data 2 . The efficacy of a vaccine against influenza, therefore, depends on the match of antigenicity between the vaccine and circulating influenza strains 3 . Additionally, other factors such as the age and immunocompetence of vaccinees, as well as preexisting amounts of antibody derived from prior infection or vaccination, contribute to mechanisms that mediate the efficacy of vaccines against influenza 1,2,4 .

Systems vaccinology has emerged as an interdisciplinary field that combines systems-wide measurements plus network and predictive modeling applied to vaccinology 5 . A systems biology approach has been used to identify early gene signatures that correlate with and can be used to predict later immune responses in humans vaccinated with the live attenuated vaccine YF-17D against yellow fever 6,7 . YF-17D is one of the most successful vaccines ever developed 8,9 it stimulates polyvalent innate immune responses 10 and adaptive immune responses 11 that can persist for decades after vaccination 11 . Although systems biology approaches have been used to predict the immunogenicity of YF-17D 6,7 , which is a live replicating virus, the extent to which such approaches can be applied to the prediction of the immunogenicity of inactivated vaccines is unknown. Furthermore, it remains unclear whether systems approaches can be used to predict the immunogenicity of recall responses. In the case of influenza, the immune response to vaccination is greatly enhanced by the past history of the vaccine recipient, both by prior infections and vaccinations. Notably, whether such approaches can provide insight into the immunological mechanisms of action of vaccines and help with the discovery of new correlates of protective immunity is untested. To address these issues, we did a series of clinical studies during the annual influenza seasons in 2007, 2008 and 2009, in which we vaccinated healthy young adults with TIV. Our goal was to undertake a detailed characterization of the innate and adaptive responses to vaccination with TIV to identify putative early signatures that correlated with or could be used to predict later immunogenicity and to obtain new insight into the mechanisms that underlie immunogenicity.

The results of our studies demonstrate that systems biology approaches can indeed be used to predict the immunogenicity of an inactivated vaccine such as TIV with up to 90% accuracy. Notably, the expression at day 3 of one of the genes in the predictive signature, encoding the kinase CaMKIV, was inversely correlated with plasma hemagglutination-inhibition (HAI) antibody titers at day 28. Vaccination of CaMKIV-deficient (Camk4 −/− ) mice with TIV induced enhanced antigen-specific antibody titers, which demonstrated an unappreciated role for CaMKIV in the regulation of antibody responses. Together our results demonstrate the utility of systems biology not only in the prediction of vaccine immunogenicity but also in offering new insight into the molecular mechanism of influenza vaccines.


Nicholas C Wu

K99/R00 Pathway to Independence Award, NIH/NIAID (2019)
Croucher Postdoctoral Fellowship, Croucher Foundation (2015-2017)
Dissertation Year Fellowship, UCLA (2014-2015)
Audree Fowler Fellowship in Protein Science, UCLA (2014)
Philip Whitcome Pre-Doctoral Fellowship, UCLA (2011-2014)

Representative Publications

Koenig PA, Das H, Liu H, Kümmerer BM, Gohr FN, Jenster LM, Schiffelers LDJ, Tesfamariam YM, Uchima M, Wuerth JD, Gatterdam K, Ruetalo N, Christensen MH, Fandrey CI, Normann S, Tödtmann JMP, Pritzl S, Hanke L, Boos J, Yuan M, Zhu X, Schmid-Burgk JL, Kato H, Schindler M, Wilson IA, Geyer M, Ludwig KU, Hällberg BM # , Wu NC # , Schmidt FI # . Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape. Science 371:eabe6230 (2021). # Co-corresponding Authors

Wu NC*, Thompson AJ*, Lee JM, Su W, Arlian BM, Xie J, Lerner RA, Yen HL, Bloom JD, Wilson IA. Different genetic barriers for resistance to HA stem antibodies in influenza H3 and H1 viruses. Science 368:1335-1340 (2020). *Equal Contributors

Yuan M*, Wu NC*, Zhu X, Lee CCD, So RTY, Lv H, Mok CKP, Wilson IA. A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV. Science 368:630-633 (2020). *Equal Contributors

Wu NC, Otwinowski J, Thompson AJ, Nycholat CM, Nourmohammad A, Wilson IA. Major antigenic site B of human influenza H3N2 viruses has an evolving local fitness landscape. Nature Communications 11:1233 (2020).

Wu NC, Lv H, Thompson AJ, Wu DC, Ng WWS, Kadam RU, Lin CW, Nycholat CM, McBride R, Liang W, Paulson JC, Mok CKP, Wilson IA. Preventing an antigenically disruptive mutation in egg-based H3N2 seasonal influenza vaccines by mutational incompatibility. Cell Host & Microbe 25:836-844.e5 (2019).


Contents

The time between exposure to the virus and development of symptoms, called the incubation period, is 1–4 days, most commonly 1–2 days. Many infections, however, are asymptomatic. [4] The onset of symptoms is sudden, and initial symptoms are predominately non-specific, including fever, chills, headaches, muscle pain or aching, a feeling of discomfort, loss of appetite, lack of energy/fatigue, and confusion. These symptoms are usually accompanied by respiratory symptoms such as a dry cough, sore or dry throat, hoarse voice, and a stuffy or runny nose. Coughing is the most common symptom. [5] Gastrointestinal symptoms may also occur, including nausea, vomiting, diarrhea, [6] and gastroenteritis, [7] especially in children. The standard influenza symptoms typically last for 2–8 days. [8]

Symptomatic infections are usually mild and limited to the upper respiratory tract, but progression to pneumonia is relatively common. Pneumonia may be caused by the primary viral infection or by a secondary bacterial infection. Primary pneumonia is characterized by rapid progression of fever, cough, labored breathing, and low oxygen levels that cause bluish skin. It is especially common among those who have an underlying cardiovascular disease such as rheumatic heart disease. Secondary pneumonia typically has a period of improvement in symptoms for 1–3 weeks [9] followed by recurrent fever, sputum production, and fluid buildup in the lungs, [5] but can also occur just a few days after influenza symptoms appear. [9] About a third of primary pneumonia cases are followed by secondary pneumonia, which is most frequently caused by the bacteria Streptococcus pneumoniae and Staphylococcus aureus. [4] [5]

Types of virus

Influenza viruses comprise four species. Each of the four species is the sole member of its own genus, and the four influenza genera comprise four of the seven genera in the family Orthomyxoviridae. They are: [5] [10]

  • Influenza A virus (IAV), genus Alphainfluenzavirus
  • Influenza B virus (IBV), genus Betainfluenzavirus
  • Influenza C virus (ICV), genus Gammainfluenzavirus
  • Influenza D virus (IDV), genus Deltainfluenzavirus

IAV is responsible for most cases of severe illness as well as seasonal epidemics and occasional pandemics. It infects people of all ages but tends to disproportionately cause severe illness in the elderly, the very young, and those who have chronic health issues. Birds are the primary reservoir of IAV, especially aquatic birds such as ducks, geese, shorebirds, and gulls, [11] [12] but the virus also circulates among mammals, including pigs, horses, and marine mammals. IAV is classified into subtypes based on the viral proteins haemagglutinin (H) and neuraminidase (N). [13] As of 2019, 18 H subtypes and 11 N subtypes have been identified. Most potential combinations have been reported in birds, but H17-18 and N10-11 have only been found in bats. Only H subtypes H1-3 and N subtypes N1-2 are known to have circulated in humans, [13] the current IAV subtypes in circulation being H1N1 and H3N2. [14] IAVs can be classified more specifically to also include natural host species, geographical origin, year of isolation, and strain number, such as H1N1/A/duck/Alberta/35/76. [5] [6]

IBV mainly infects humans but has been identified in seals, horses, dogs, and pigs. [13] IBV does not have subtypes like IAV but has two antigenically distinct lineages, termed the B/Victoria/2/1987-like and B/Yamagata/16/1988-like lineages, [5] or simply (B/)Victoria(-like) and (B/)Yamagata(-like). [13] [14] Both lineages are in circulation in humans, [5] disproportionately affecting children. [6] IBVs contribute to seasonal epidemics alongside IAVs but have never been associated with a pandemic. [13]

ICV, like IBV, is primarily found in humans, though it also has been detected in pigs, feral dogs, dromedary camels, cattle, and dogs. [7] [13] ICV infection primarily affects children and is usually asymptomatic [5] [6] or has mild cold-like symptoms, though more severe symptoms such as gastroenteritis and pneumonia can occur. [7] Unlike IAV and IBV, ICV has not been a major focus of research pertaining to antiviral drugs, vaccines, and other measures against influenza. [13] ICV is subclassified into six genetic/antigenic lineages. [7] [15]

IDV has been isolated from pigs and cattle, the latter being the natural reservoir. Infection has also been observed in humans, horses, dromedary camels, and small ruminants such as goats and sheep. [13] [15] IDV is distantly related to ICV. While cattle workers have occasionally tested positive to prior IDV infection, it is not known to cause disease in humans. [5] [6] [7] ICV and IDV experience a slower rate of antigenic evolution than IAV and IBV. Because of this antigenic stability, relatively few novel lineages emerge. [15]

Genome and structure

Influenza viruses have a negative-sense, single-stranded RNA genome that is segmented. The negative sense of the genome means it can be used as a template to synthesize messenger RNA (mRNA). [4] IAV and IBV have eight genome segments that encode 10 major proteins. ICV and IDV have seven genome segments that encode nine major proteins. [7] Three segments encode three subunits of an RNA-dependent RNA polymerase (RdRp) complex: PB1, a transcriptase, PB2, which recognizes 5' caps, and PA (P3 for ICV and IDV), an endonuclease. [16] The matrix protein (M1) and membrane protein (M2) share a segment, as do the non-strucutral protein (NS1) and the nuclear export protein (NEP). [5] For IAV and IBV, hemagglutinin (HA) and neuraminidase (NA) are encoded on one segment each, whereas ICV and IDV encode a hemagglutinin-esterase fusion (HEF) protein on one segment that merges the functions of HA and NA. The final genome segment encodes the viral nucleoprotein (NP). [16] Influenza viruses also encode various accessory proteins, such as PB1-F2 and PA-X, that are expressed through alternative open reading frames [5] [17] and which are important in host defense suppression, virulence, and pathogenicity. [18]

The virus particle, called a virion, is spherical, filamentous, or pleomorphic in shape and 80–120 nanometers (nm) in diameter. [16] The virion consists of each segment of the genome bound to nucleoproteins in separate ribonucleoprotein (RNP) complexes for each segment, all of which are surrounded by a lipid bilayer membrane called the viral envelope. There is a copy of the RdRp, all subunits included, bound to each RNP. The envelope is reinforced structurally by matrix proteins on the interior that enclose the RNPs, [19] and the envelope contains HA and NA (or HEF [15] ) proteins extending outward from the exterior surface of the envelope. HA and HEF [15] proteins have a distinct "head" and "stalk" structure. M2 proteins form proton ion channels through the viral envelope that are required for viral entry and exit. IBVs contain a surface protein named NB that is anchored in the envelope, but its function is unknown. [5] NS1, NEP, PB1-F2, and PA-X are only expressed in host cells and are not found in the virion. [17]

Life cycle

The viral life cycle begins by binding to a target cell. Binding is mediated by the viral HA proteins on the surface of the evelope, which bind to cells that contain sialic acid receptors on the surface of the cell membrane. [5] [11] [19] For N1 subtypes with the "G147R" mutation and N2 subtypes, the NA protein can initiate entry. Prior to binding, NA proteins promote access to target cells by degrading mucous, which helps to remove extracellular decoy receptors that would impede access to target cells. [19] After binding, the virus is internalized into the cell by an endosome that contains the virion inside it. The endosome is acidified by cellular vATPase [17] to have lower pH, which triggers a conformational change in HA that allows fusion of the viral envelope with the endosomal membrane. [18] At the same time, hydrogen ions diffuse into the virion through M2 ion channels, disrupting internal protein-protein interactions to release RNPs into the host cell's cytosol. The M1 protein shell surrounding RNPs is degraded, fully uncoating RNPs in the cytosol. [17] [19]

RNPs are then imported into the nucleus with the help of viral localization signals. There, the viral RNA polymerase transcribes mRNA using the genomic negative-sense strand as a template. The polymerase snatches 5' caps for viral mRNA from cellular RNA to prime mRNA synthesis and the 3'-end of mRNA is polyadenylated at the end of transcription. [16] Once viral mRNA is transcribed, it is exported out of the nucleus and translated by host ribosomes in a cap-dependent manner to synthesize viral proteins. [17] RdRp also synthesizes complementary positive-sense strands of the viral genome in a complementary RNP complex which are then used as templates by viral polymerases to synthesize copies of the negative-sense genome. [5] [19] During these processes, RdRps of avian influenza viruses (AIVs) function optimally at a higher temperature than mammalian influenza viruses. [8]

Newly synthesized viral polymerase subunits and NP proteins are imported to the nucleus to further increase the rate of viral replication and form RNPs. [16] HA, NA, and M2 proteins are trafficked with the aid of M1 and NEP proteins [18] to the cell membrane through the Golgi apparatus [16] and inserted into the cell's membrane. Viral non-structural proteins including NS1, PB1-F2, and PA-X regulate host cellular processes to disable antiviral responses. [5] [18] [19] PB1-F2 aso interacts with PB1 to keep polymerases in the nucleus longer. [12] M1 and NEP proteins localize to the nucleus during the later stages of infection, bind to viral RNPs and mediate their export to the cytoplasm where they migrate to the cell membrane with the aid of recycled endosomes and are bundled into the segments of the genome. [5] [19]

Progenic viruses leave the cell by budding from the cell membrane, which is initiated by the accumulation of M1 proteins at the cytoplasmic side of the membrane. The viral genome is incorporated inside a viral envelope derived from portions of the cell membrane that have HA, NA, and M2 proteins. At the end of budding, HA proteins remain attached to cellular sialic acid until they are cleaved by the sialidase activity of NA proteins. The virion is then released from the cell. The sialidase activity of NA also cleaves any sialic acid residues from the viral surface, which helps prevent newly assembled viruses from aggregating near the cell surface and improving infectivity. [5] [19] Similar to other aspects of influenza replication, optimal NA activity is temperature- and pH-dependent. [8] Ultimately, presence of large quantities of viral RNA in the cell triggers apoptosis, i.e. programmed cell death, which is initiated by cellular factors to restrict viral replication. [17]

Antigenic drift and shift

Two key processes that influenza viruses evolve through are antigenic drift and antigenic shift. Antigenic drift is when an influenza virus's antigens change due to the gradual accumulation of mutations in the antigen's (HA or NA) gene. [11] This can occur in response to evolutionary pressure exerted by the host immune response. Antigenic drift is especially common for the HA protein, in which just a few amino acid changes in the head region can constitute antigenic drift. [14] [15] The result is the production of novel strains that can evade pre-existing antibody-mediated immunity. [5] [6] Antigenic drift occurs in all influenza species but is slower in B than A and slowest in C and D. [15] Antigenic drift is a major cause of seasonal influenza, [20] and requires that flu vaccines be updated annually. HA is the main component of inactivated vaccines, so surveillance monitors antigenic drift of this antigen among circulating strains. Antigenic evolution of influenza viruses of humans appears to be faster than influenza viruses in swine and equines. In wild birds, within-subtype antigenic variation appears to be limited but has been observed in poultry. [5] [6]

Antigenic shift is a sudden, drastic change in an influenza virus's antigen, usually HA. During antigenic shift, antigenically different strains that infect the same cell can reassort genome segments with each other, producing hybrid progeny. Since all influenza viruses have segmented genomes, all are capable of reassortment. [7] [15] Antigenic shift, however, only occurs among influenza viruses of the same genus [16] and most commonly occurs among IAVs. In particular, reassortment is very common in AIVs, creating a large diversity of influenza viruses in birds, but is uncommon in human, equine, and canine lineages. [21] Pigs, bats, and quails have receptors for both mammalian and avian IAVs, so they are potential "mixing vessels" for reassortment. [13] If an animal strain reassorts with a human strain, [14] then a novel strain can emerge that is capable of human-to-human transmission. This has caused pandemics, but only a limited number have occurred, so it is difficult to predict when the next will happen. [5] [6]

Transmission

People who are infected can transmit influenza viruses through breathing, talking, coughing, and sneezing, which spread respiratory droplets and aerosols that contain virus particles into the air. A person susceptible to infection can then contract influenza by coming into contact with these particles. [9] [22] Respiratory droplets are relatively large and travel less than two meters before falling onto nearby surfaces. Aerosols are smaller and remain suspended in the air longer, so they take longer to settle and can travel further than respiratory droplets. [22] [23] Inhalation of aerosols can lead to infection, [24] but most transmission is in the area about two meters around an infected person via respiratory droplets [4] that come into contact with mucosa of the upper respiratory tract. [24] Transmission through contact with a person, bodily fluids, or intermediate objects (fomites) can also occur, such as through contaminated hands and surfaces [4] [22] since influenza viruses can survive for hours on non-porous surfaces. [23] If one's hands are contaminated, then touching one's face can cause infection. [25]

Influenza is usually transmissible from one day before the onset of symptoms to 5–7 days after. [6] In healthy adults, the virus is shed for up to 3–5 days. In children and the immunocompromised, the virus may be transmissible for several weeks. [4] Children ages 2–17 are considered to be the primary and most efficient spreaders of influenza. [5] [6] Children who have not had multiple prior exposures to influenza viruses shed the virus at greater quantities and for a longer duration than other children. [5] People who are at risk of exposure to influenza include health care workers, social care workers, and those who live with or care for people vulnerable to influenza. In long-term care facilities, the flu can spready rapidly after it is introduced. [6] A variety of factors likely encourage influenza transmission, including lower temperature, lower absolute and relative humidity, less ultraviolet radiation from the Sun, [24] [26] and crowding. [22] Influenza viruses that infect the upper respiratory tract like H1N1 tend to be more mild but more transmissible, whereas those that infect the lower respiratory tract like H5N1 tend to cause more severe illness but are less contagious. [4]

Pathophysiology

In humans, influenza viruses first cause infection by infecting epithelial cells in the respiratory tract. Illness during infection is primarily the result of lung inflammation and compromise caused by epithelial cell infection and death, combined with inflammation caused by the immune system's response to infection. Non-respiratory organs can become involved, but the mechanisms by which influenza is involved in these cases is unknown. Severe respiratory illness can be caused by multiple, non-exclusive mechanisms, including obstruction of the airways, loss of alveolar structure, loss of lung epithelial integrity due to epithelial cell infection and death, and degradation of the extracellular matrix that maintains lung structure. In particular, alveolar cell infection appears to drive severe symptoms since this results in impaired gas exchange and enables viruses to infect endothelial cells, which produce large quantities of pro-inflammatory cytokines. [9]

Pneumonia caused by influenza viruses is characterized by high levels of viral replication in the lower respiratory tract, accompanied by a strong pro-inflammatory response called a cytokine storm. [5] Infection with H5N1 or H7N9 especially produces high levels of pro-inflammatory cytokines. [11] In bacterial infections, early depletion of macrophages during influenza creates a favorable environment in the lungs for bacterial growth since these white blood cells are important in responding to bacterial infection. Host mechanisms to encourage tissue repair may inadvertently allow bacterial infection. Infection also induces production of systemic glucocorticoids that can reduce inflammation to preserve tissue integrity but allow increased bacterial growth. [9]

The pathophysiology of influenza is significantly influenced by which receptors influenza viruses bind to during entry into cells. Mammalian influenza viruses preferentially bind to sialic acids connected to the rest of the oligosaccharide by an α-2,6 link, most commonly found in various respiratory cells, [5] [11] [19] such as respiratory and retinal epithelial cells. [17] AIVs prefer sialic acids with an α-2,3 linkage, which are most common in birds in gastrointestinal epithelial cells [5] [11] [19] and in humans in the lower respiratory tract. [27] Furthermore, cleavage of the HA protein into HA1, the binding subunit, and HA2, the fusion subunit, is performed by different proteases, affecting which cells can be infected. For mammalian influenza viruses and low pathogenic AIVs, cleavage is extracellular, which limits infection to cells that have the appropriate proteases, whereas for highly pathogenic AIVs, cleavage is intracellular and performed by ubiqutious proteases, which allows for infection of a greater variety of cells, thereby contributing to more severe disease. [5] [21] [28]

Immunology

Cells possess sensors to detect viral RNA, which can then induce interferon production. Interferons mediate expression of antiviral proteins and proteins that recruit immune cells to the infection site, and they also notify nearby uninfected cells of infection. Some infected cells release pro-inflammatory cytokines that recruit immune cells to the site of infection. Immune cells control viral infection by killing infected cells and phagocytizing viral particles and apoptotic cells. An exacerbated immune response, however, can harm the host organism through a cytokine storm. [5] [8] [17] To counter the immune response, influenza viruses encode various non-structural proteins, including NS1, NEP, PB1-F2, and PA-X, that are involved in curtailing the host immune response by suppressing interferon production and host gene expression. [5] [18]

B cells, a type of white blood cell, produce antibodies that bind to influenza antigens HA and NA (or HEF [15] ) and other proteins to a lesser degree. Once bound to these proteins, antibodies block virions from binding to cellular receptors, neutralizing the virus. The antibody response to influenza in humans is typically robust and long-lasting, [5] especially for ICV and IDV since HEF is antigenically stable. [15] In other words, people exposed to a certain strain in childhood still possess antibodies to that strain at a reasonable level later in life, which can provide some protection to related strains. [5] There is, however, an "original antigenic sin", in which the first HA subtype a person is exposed to influences the antibody-based immune response to future infections and vaccines. [14]

Vaccination

Annual vaccination is the primary and most effective way to prevent influenza and influenza-associated complications, especially for high-risk groups. [4] [5] [29] Vaccines against the flu are trivalent or quadrivalent, providing protection against an H1N1 strain, an H3N2 strain, and one or two IBV strains corresponding to the two IBV lineages. [4] [14] Two types of vaccines are in use: inactivated vaccines that contain "killed" (i.e. inactivated) viruses and live attenuated influenza vaccines (LAIVs) that contain weakened viruses. [5] There are three types of inactivated vaccines: whole virus, split virus, in which the virus is disrupted by a detergent, and subunit, which only contains the viral antigens HA and NA. [30] Most flu vaccines are inactivated and administered via intramuscular injection. LAIVs are sprayed into the nasal cavity. [5]

Vaccination recommendations vary from country to country. It some countries, it is recommended that all people above a certain age, such as 6 months, [29] be vaccinated, whereas in others it is recommended for people who belong to at-risk groups, such as pregnant women, young children (excluding newborns), the elderly, people with chronic medical conditions, health care workers, [5] people who come into contact with high-risk people, and people who transmit the virus easily. [6] Young infants cannot receive flu vaccines for safety reasons, but they can inherit passive immunity from their mother if inactivated vaccines are administered to the mother during pregnancy. [31] Influenza vaccination also helps to reduce the probability of reassortment. [8]

The effectiveness of seasonal flu vaccines varies significantly, with an estimated average efficacy of 50–60%, [14] depending on vaccine strain, age, prior immunity, and immune function, so vaccinated people can still contract influenza. [29] The effectiveness of flu vaccines is considered to be suboptimal, particularly among the elderly, [5] but vaccination is still beneficial in reducing the mortality rate and hospitalization rate due to influenza as well as duration of hospitalization. [29] [32] Vaccination of school-age children has shown to provide indirect protection for other age groups. LAIVs are recommended for children based on superior efficacy, especially for children under 6, and greater immunity against non-vaccine strains when compared to inactivated vaccines. [6] [31]

Common side effects of vaccination include local injection-site reactions and cold-like symptoms. Fever, malaise, and myalgia are less common. Flu vaccines are contraindicated for people who have experienced a severe allergic reaction in response to a flu vaccine or to any component of the vaccine. LAIVs are not given to children or adolescents with severe immunodeficiency or to those who are using salicylate treatments because of the risk of developing Reye syndrome. [6] LAIVs are also not recommended for children under the age of 2, [31] pregnant women, and adults with immunosuppression. Inactivated flu vaccines cannot cause influenza and are regarded as safe during pregnancy. [6]

In general, influenza vaccines are only effective if there is an antigenic match between vaccine strains and circulating strains. [4] [14] Additionally, most commercially available flu vaccines are manufactured by propagation of influenza viruses in embryonated chicken eggs, taking 6–8 months. [14] Flu seasons are different in the northern and southern hemisphere, so the WHO meets twice a year, one for each hemisphere, to discuss which strains should be included in flu vaccines based on observation from HA inhibition assays. [4] [19] Other manufacturing methods include an MDCK cell culture-based inactivated vaccine and a recombinant subunit vaccine manufactured from baculovirus overexpression in insect cells. [14] [33]

Antiviral chemoprophylaxis

Influenza can be prevented or reduced in severity by post-exposure prophylaxis with the antiviral drugs oseltamivir, which can be taken orally by those at least three months old, and zanamivir, which can be inhaled by those above seven years of age. Chemoprophylaxis is most useful for individuals at high-risk of developing complications and those who cannot receive the flu vaccine due to contraindiciations or lack of effectiveness. [4] Post-exposure chemoprophylaxis is only recommended if oseltamivir is taken within 48 hours of contact with a confirmed or suspected influenza case and zanamivir within 36 hours. [4] [6] It is recommended that it be offered to people who have yet to receive a vaccine for the current flu season, who have been vaccinated less than two week since contact, if there is a significant mismatch between vaccine and circulating strains, or during an outbreak in a closed setting regardless of vaccination history. [6]

Infection control

Hand hygiene is imoprtant in reducing the spread of influenza. This includes measures such as frequent hand washing with soap and water, using alcohol-based hand sanitizers, and not touching one's eyes, nose, and mouth with one's hands. Covering one's nose and mouth when coughing or sneezing is important. [34] Other methods to limit influenza transmission include staying home when sick, [5] avoiding contact with others until one day after symptoms end, [6] and disinfecting surfaces likely to be contaminated by the virus, such as doorknobs. [5] Health education through media and posters is often used to remind people of the aforementioned etiquette and hygiene. [4]

There is uncertainty about the use of masks since research thus far has not shown a significant reduction in seasonal influenza with mask usage. Likewise, the effectiveness of screening at points of entry into countries is not well researched. [34] Social distancing measures such as school closures, avoiding contact with infected people via isolation or quarantine, and limiting mass gatherings may reduce transmission, [5] [34] but these measures are often expensive, unpopular, and difficult to implement. Consequently, the commonly recommended methods of infection control are respiratory etiquette, hand hygiene, and mask wearing, which are inexpensive and easy to perform. Pharmaceutical measures are effective but may not be available in the early stages of an outbreak. [35]

In health care settings, infected individuals may be cohorted or assigned to individual rooms. Protective clothing such as masks, gloves, and gowns is recommended when coming into contact with infected individuals if there is a risk of exposure to infected bodily fluids. Keeping patients in negative pressure rooms and avoiding aerosol-producing activities may help, [4] but special air handling and ventilation systems are not considered necessary to prevent the spread of influenza in the air. [23] In residential homes, new admissions may need to be closed until the spread of influenza is controlled. When discharging patients to care homes, it is important to take care if there is a known influenza outbreak. [6]

Since influenza viruses circulate in animals such as birds and pigs, prevention of transmission from these animals is important. Water treatment, indoor raising of animals, quarantining sick animals, vaccination, and biosecurity are the primary measures used. Placing poultry houses and piggeries on high ground away from high-density farms, backyard farms, live poultry markets, and bodies of water helps to minimize contact with wild birds. [5] Closure of live poultry markets appears to the most effective measure [11] and has shown to be effective at controlling the spread of H5N1, H7N9, and H9N2. [12] Other biosecurity measures include cleaning and disinfecting facilities and vehicles, banning visits to poultry farms, not bringing birds intended for slaughter back to farms, [36] changing clothes, disinfecting foot baths, and treating food and water. [5]

If live poultry markets are not closed, then "clean days" when unsold poultry is removed and facilities are disinfected and "no carry-over" policies to eliminate infectious material before new poultry arrive can be used to reduce the spread of influenza viruses. If a novel influenza viruses has breached the aforementioned biosecurity measures, then rapid detection to stamp it out via quarantining, decontamination, and culling may be necessary to prevent the virus from becoming endemic. [5] Vaccines exist for avian H5, H7, and H9 subtypes that are used in some countries. [11] In China, for example, vaccination of domestic birds against H7N9 successfully limited its spread, indicating that vaccination may be an effective strategy [21] if used in combination with other measures to limit transmission. [5] In pigs and horses, management of influenza is dependent on vaccination with biosecurity. [5]

Diagnosis based on symptoms is fairly accurate in otherwise healthy people during seasonal epidemics and should be suspected in cases of pneumonia, acute respiratory distress syndrome (ARDS), sepsis, or if encephalitis, myocarditis, or breaking down of muscle tissue occur. [9] Because influenza is similar to other viral respiratory tract illnesses, laboratory diagnosis is necessary for confirmation. Common ways of collecting samples for testing include nasal and throat swabs. [5] Samples may be taken from the lower respiratory tract if infection has cleared the upper but not lower respiratory tract. Influenza testing is recommended for anyone hospitalized with symptoms resembling influenza during flu season or who is connected to an influenza case. For severe cases, earlier diagnosis improves patient outcome. [29] Diagnostic methods that can identify influenza include viral cultures, antibody- and antigen-detecting tests, and nucleic acid-based tests. [37]

Viruses can be grown in a culture of mammalian cells or embryonated eggs for 3–10 days to monitor cytopathic effect. Final confirmation can then be done via antibody staining, hemadsorption using red blood cells, or immunofluorescence microscopy. Shell vial cultures, which can identify infection via immunostaining before a cytopathic effect appears, are more sensitive than traditional cultures with results in 1–3 days. [5] [29] [37] Cultures can be used to characterize novel viruses, observe sensitivity to antiviral drugs, and monitor antigenic drift, but they are relatively slow and require specialized skills and equipment. [5]

Serological assays can be used to detect an antibody response to influenza after natural infection or vaccination. Common serological assays include hemagglutination inhibition assays that detect HA-specific antibodies, virus neutralization assays that check whether antibodies have neutralized the virus, and enzyme-linked immunoabsorbant assays. These methods tend to be relatively inexpensive and fast but are less reliable than nucleic-acid based tests. [5] [37]

Direct fluorescent or immunofluorescent antibody (DFA/IFA) tests involve staining respiratory epithelial cells in samples with fluorescently-labeled influenza-specific antibodies, followed by examination under a fluorescent microscope. They can differentiate between IAV and IBV but can't subtype IAV. [37] Rapid influenza diagnostic tests (RIDTs) are a simple way of obtaining assay results, are low cost, and produce results quickly, at less than 30 minutes, so they are commonly used, but they can't distinguish between IAV and IBV or between IAV subtypes and are not as sensitive as nucleic-acid based tests. [5] [37]

Nucleic acid-based tests (NATs) amplify and detect viral nucleic acid. Most of these tests take a few hours, [37] but rapid molecular assays are as fast as RIDTs. [29] Among NATs, reverse transcription polymerase chain reaction (RT-PCR) is the most traditional and considered the gold standard for diagnosing influenza [37] because it is fast and can subtype IAV, but it is relatively expensive and more prone to false-positives than cultures. [5] Other NATs that have been used include loop-mediated isothermal amplification-based assays, simple amplification-based assays, and nucleic acid sequence-based amplification. Nucleic acid sequencing methods can identify infection by obtaining the nucleic acid sequence of viral samples to identify the virus and antiviral drug resistance. The traditional method is Sanger sequencing, but it has been largely replaced by next-generation methods that have greater sequencing speed and throughput. [37]

Treatment of influenza in cases of mild or moderate illness is supportive and includes anti-fever medications such as acetaminophen and ibuprofen, [38] adequate fluid intake to avoid dehydration, and resting at home. [6] Cough drops and throat sprays may be beneficial for sore throat. It is recommended to avoid alcohol and tobacco use while sick with the flu. [38] Aspirin should not be used to treat influenza in children due to an elevated risk of developing Reye syndrome. [39] Corticosteroids likewise are not recommended except when treating septic shock or an underyling medical condition, such as chronic obstructive pulmonary disease or asthma exacerbation, since they are associated with increased mortality. [29] If a secondary bacterial infection occurs, then treatment with antibiotics may be necessary. [6]

Antivirals

Antiviral drugs [8]
Drug Route of administration Approved age of use
Oseltamivir Oral At least two weeks old
Zanamivir Inhalation At least five years old
Peramivir Intravenous injection At least 18 years old
Laninamivir Inhalation [5] 40 milligrams (mg) dose for people at least 10 years old,
20 mg for those under 10 [40]
Baloxavir marboxil Oral [23] At least 12 years old [29]

Antiviral drugs are primarily used to treat severely ill patients, especially those with compromised immune systems. Antivirals are most effective when started in the first 48 hours after symptoms appear. Later administration may still be beneficial for those who have underlying immune defects, those with more severe symptoms, or those who have a higher risk of developing complications if these individuals are still shedding the virus. Antiviral treatment is also recommended if a person is hospitalized with suspected influenza instead of waiting for test results to return and if symptoms are worsening. [5] [29] Most antiviral drugs against influenza fall into two categories: neuraminidase (NA) inhibitors and M2 inhibitors. [8] Baloxavir marboxil is a notable exception, which targets the endonuclease activity of the viral RNA polymerase and can be used as an alternative to NA and M2 inhibitors for IAV and IBV. [4] [11] [23]

NA inhibitors target the enzymatic activity of NA receptors, mimicking the binding of sialic acid in the active site of NA on IAV and IBV virions [5] so that viral release from infected cells and the rate of viral replication are impaired. [6] NA inhibitors include oseltamivir, which is consumed orally in a prodrug form and converted to its active form in the liver, and zanamivir, which is a powder that is inhaled nasally. Oseltamivir and zanamivir are effective for prophylaxis and post-exposure prophylaxis, and research overall indicates that NA inhibitors are effective at reducing rates of complications, hospitalization, and mortality [5] and the duration of illness. [8] [29] [23] Additionally, the earlier NA inhibitors are provided, the better the outcome, [23] though late administration can still be beneficial in severe cases. [5] [29] Other NA inhibitors include laninamivir [5] and peramivir, the latter of which can be used as an alternative to oseltamivir for people who cannot tolerate or absorb it. [29]

The adamantanes amantadine and rimantadine are orally administered drugs that block the influenza virus's M2 ion channel, [5] preventing viral uncoating. [23] These drugs are only functional against IAV [29] but are no longer recommended for use because of widespread resistance to them among IAVs. [23] Adamantane resistance first emerged in H3N2 in 2003, becoming worldwide by 2008. Oseltamivir resistance is no longer widespread because the 2009 pandemic H1N1 strain (H1N1 pdm09), which is resistant to adamantanes, seemingly replaced resistant strains in circulation. Since the 2009 pandemic, oseltamivir resistance has mainly been observed in patients undergoing therapy, [5] especially the immunocompromised and young children. [23] Oseltamivir resistance is usually reported in H1N1, but has been reported in H3N2 and IBVs less commonly. [5] Because of this, oseltamivir is recommended as the first drug of choice for immunocompetent people, whereas for the immunocompromised, oseltamivir is recommended against H3N2 and IBV and zanamivir against H1N1 pdm09. Zanamivir resistance is observed less frequently, and resistance to peramivir and baloxavir marboxil is possible. [23]

In healthy individuals, influenza infection is usually self-limiting and rarely fatal. [4] [6] Symptoms usually last for 2–8 days. [8] Influenza can cause people to miss work or school, and it is associated with decreased job performance and, in older adults, reduced independence. Fatigue and malaise may last for several weeks after recovery, and healthy adults may experience pulmonary abnormalities that can take several weeks to resolve. Complications and mortality primarily occur in high-risk populations and those who are hospitalized. Severe disease and mortality are usually attributable to pneumonia from the primary viral infection or a secondary bacterial infection, [5] [6] which can progress to ARDS. [8]

Other respiratory complications that may occur include sinusitis, bronchitis, bronchiolitis, excess fluid buildup in the lings, and exercerbation of chronic bronchitis and asthma. Middle ear infection and croup may occur, most commonly in children. [4] [5] Secondary S. aureus infection has been observed, primarily in children, to cause toxic shock syndrome after influenza, with hypotension, fever, and reddening and peeling of the skin. [5] Complications affecting the cardiovascular system are rare and include pericarditis, fulminant myocarditis with a fast, slow, or irregular heartbeat, and exacerbation of pre-existing cardiovascular disease. [4] [6] Inflammation or swelling of muscles accompanied by muscle tissue breaking down occurs rarely, usually in children, which presents as extreme tenderness and muscle pain in the legs and a reluctance to walk for 2–3 days. [5] [6] [9]

Influenza can affect pregnancy, including causing smaller neonatal size, increased risk of premature birth, and an increased risk of child death shortly before or after birth. [6] Neurological complications have been associated with influenza on rare ocassions, including aseptic meningitis, encephalitis, disseminated encephalomyelitis, transverse myelitis, and Guillain–Barré syndrome. [9] Additionally, febrile seizures and Reye syndrome can occur, most commonly in children. [5] [6] Influenza-associated encephalopathy can occur directly from central nervous system infection from the presence of the virus in blood and presents as suddent onset of fever with convulsions, followed by rapid progression to coma. [4] An atypical form of encephalitis called encephalitis lethargica, characterized by headache, drowsiness, and coma, may rarely occur sometime after infection. [5] In survivors of influenza-associated encephalopathy, neurological defects may occur. [4] Primarily in children, in severe cases the immune system may rarely dramatically overproduce white blood cells that release cytokines, causing severe inflammation. [4]

People who are at least 65 years of age, [6] due to a weakened immune system from aging or a chronic illness, are a high-risk group for developing complications, as are children less than one year of age and children who have not been previously exposed to influenza viruses multiple times. Pregnant women are at an elevated risk, which increases by trimester [5] and lasts up to two weeks after childbirth. [6] [29] Obesity, in particular a body mass index greater than 35–40, is associated with greater amounts of viral replication, increased severity of secondary bacterial infection, and reduced vaccination efficacy. People who have underlying health conditions are also considered at-risk, including those who have congenital or chronic heart problems or lung (e.g. asthma), kidney, liver, blood, neurological, or metabolic (e.g. diabetes) disorders, [4] [5] [6] as are people who are immunocompromised from chemotherapy, asplenia, prolonged steroid treatment, splenic dysfunction, or HIV infection. [6] Current or past tobacco use also places a person at risk. [29] The role of genetics in influenza is not well researched, [5] but it may be a factor in influenza mortality. [8]

Influenza is typically characterized by seasonal epidemics and sporadic pandemics. Most of the burden of influenza is a result of flu seasons caused by IAV and IBV. Among IAV subtypes, H1N1 and H3N2 currently circulate in humans and are responsible for seasonal influenza. Cases disproportionately occur in children, but most severe causes are among the elderly, the very young, [5] and the immunocompromised. [23] In a typical year, influenza viruses infect 5–15% of the global population, [19] [37] causing 3–5 million cases of severe illness annually [5] [14] and accounting for 290,000–650,000 deaths each year due to respiratory illness. [19] [23] [42] 5–10% of adults and 20–30% of children contract influenza each year. [13] The reported number of influenza cases is usually much lower than the actual number of cases. [5] [31]

During seasonal epidemics, it is estimated that about 80% of otherwise healthy people who have a cough or sore throat have the flu. [5] Approximately 30–40% of people hospitalized for influenza develop pneumonia, and about 5% of all severe pneumonia cases in hospitals are due to influenza, which is also the most common cause of ARDS in adults. In children, influenza is one of the two most common causes of ARDS, the other being the respiratory syncytial virus. [9] About 3–5% of children each year develop otitis media due to influenza. [4] Adults who develop organ failure from influenza and children who have PIM scores and acute renal failure have higher rates of mortality. [9] During seasonal influenza, mortality is concentrated in the very young and the elderly, whereas during flu pandemics, young adults are often affected at a high rate. [8]

In temperate regions, the number of influenza cases varies from season to season. Lower vitamin D levels, presumably due to less sunlight, [26] lower humidity, lower temperature, and minor changes in virus proteins caused by antigenic drift contribute to annual epidemics that peak during the winter season. In the northern hemisphere, this is from October to May (more narrowly December to April [8] ), and in the southern hemisphere, this is from May to October (more narrowly June to September [8] ). There are therefore two distinct influenza seasons every year in temperate regions, one in the northern hemisphere and one in the southern hemisphere. [5] [6] [14] In tropical and subtropical regions, seasonality is more complex and appears to be affected by various climatic factors such as minimum temperature, hours of sunshine, maximum rainfall, and high humidity. [5] [43] Influenza may therefore occur year-round in these regions. [8] Influenza epidemics in modern times have the tendency to start in the eastern or southern hemisphere, [43] with Asia being a key reservoir of influenza viruses. [8]

IAV and IBV co-circulate, so the two have the same patterns of transmission. [5] The seasonality of ICV, however, is poorly understood. ICV infection is most common in children under the age of 2, and by adulthood most people have been exposed to it. ICV-associated hospitalization most commonly occurs in children under the age of 3 and is frequently accompanied by co-infection with another virus or a bacterium, which may increase the severity of disease. When considering all hospitalizations for respiratory illness among young children, ICV appears to account for only a small percentage of such cases. Large outbreaks of ICV infection can occur, so incidence varies significantly. [7]

Outbreaks of influenza caused by novel influenza viruses are common. [16] Depending on the level of pre-existing immunity in the population, novel influenza viruses can spready rapidly and cause pandemics with millions of deaths. These pandemics, in contrast to seasonal influenza, are caused by antigenic shifts involving animal influenza viruses. To date, all known flu pandemics have been caused by IAVs, and they follow the same pattern of spreading from an origin point to the rest of the world over the course of multiple waves in a year. [5] [6] [29] Pandemic strains tend to be associated with higher rates of pneumonia in otherwise healthy individuals. [9] Generally after each influenza pandemic, the pandemic strain continues to circulate as the cause of seasonal influenza, replacing prior strains. [5] From 1700 to 1889, influenza pandemics occurred about once every 50–60 years. Since then, pandemics have occurred about once every 10–50 years, so they may be getting more frequent over time. [43]

It is impossible to know when an influenza virus first infected humans or when the first influenza pandemic occurred. [45] Possibly the first influenza epidemic occurred around 6,000 BC in China, [46] and possible descriptions of influenza exist in Greek writings from the 5th century BC. [43] [47] In both 1173–1174 and 1387, epidemics occurred across Europe that were named "influenza". Whether these epidemics and others were caused by influenza is unclear since there was no consistent naming pattern for epidemic respiratory diseases at that time, and "influenza" didn't become completely attached to respiratory disease until centuries later. [48] Influenza may have been brought to the Americas as early as 1493, when an epidemic disease resembling influenza killed most of the population of the Antilles. [49] [50]

The first convincing record of an influenza pandemic was chronicled in 1510 it began in East Asia before spreading to North Africa and then Europe. [45] Following the pandemic, seasonal influenza occurred, with subsequent pandemics in 1557 and 1580. [48] The flu pandemic in 1557 was potentially the first time influenza was connected to miscarriage and death of pregnant women. [51] The 1580 flu pandemic originated in Asia during summer, spread to Africa, then Europe, and finally America. [43] By the end of the 16th century, influenza was likely beginning to become understood as a specific, recognizable disease with epidemic and endemic forms. [48] In 1648, it was discovered that horses also experience influenza. [45]

Influenza data after 1700 is more informative, so it is easier to identify flu pandemics after this point, each of which incrementally increased understanding of influenza. [52] The first flu pandemic of the 18th century started in 1729 in Russia in spring, spreading worldwide over the course of three years with distinct waves, the later ones being more lethal. The second flu pandemic of the 18th century was in 1781–1782, starting in China in autumn. [43] From this pandemic, influenza became associated with sudden outbreaks of febrile illness. [52] The next flu pandemic was from 1830 to 1833, beginning in China in winter. This pandemic had a high attack rate, but the mortality rate was low. [20] [43]

A minor influenza pandemic occurred from 1847 to 1851 at the same time as the third cholera pandemic and was the first flu pandemic to occur with vital statistics being recorded, so influenza mortality was clearly recorded for the first time. [52] Highly pathogenic avian influenza was recognized in 1878 [52] and was soon linked to transmission to humans. [45] By the time of the 1889 pandemic, which may have been caused by an H2N2 strain, [53] the flu had become an easily recognizable disease. [45]

Initially, the microbial agent responsible for influenza was incorrently identified in 1892 by R. F. J. Pfeiffer as the bacteria species Haemophilus influenzae, which retains "influenza" in its name. [45] [52] In the following years, the field of virology began to form as viruses were identified as the cause of many diseases. From 1901 to 1903, Italian and Austrian researchers were able to show that avian influenza, then called "fowl plague", [21] was caused by a microscopic agent smaller than bacteria by using filters with pores too small for bacteria to pass through. The fundamental differences between viruses and bacteria, however, were not yet fully understood. [52]

From 1918 to 1920, the Spanish flu pandemic became the most devastating influenza pandemic and one of the deadliest pandemics in history. The pandemic, probably caused by H1N1, likely began in the USA before spreading worldwide by soldiers during and after the First World War. The initial wave in the first half of 1918 was relatively minor and resembled past flu pandemics, but the second wave later that year had a much higher mortality rate, [43] accounting for most deaths. A third wave with lower mortality occurred in many places a few months after the second. [20] By the end of 1920, it is estimated that about a third [8] to half of all people in the world had been infected, with tens of millions of deaths, disproportionately young adults. [43] During the 1918 pandemic, the respiratory route of transmission was clearly identified [20] and influenza was shown to be caused by a "filter passer", not a bacterium, but there remained a lack of agreement about influenza's cause for another decade and research on influenza declined. [52] After the pandemic, H1N1 circulated in humans in seasonal form [5] up until the next pandemic. [52]

In 1931, Richard Shope published three papers identifying a virus as the cause of swine influenza, a then newly recognized disease among pigs that was first characterized during the second wave of the 1918 pandemic. [51] [52] Shope's research reinvigorated research on human influenza, and many advances in virology, serology, immunology, experimental animal models, vaccinology, and immunotherapy have since arisen from influenza research. [52] Just two years after influenza viruses were discovered, in 1933, IAV was identified as the agent responsible for human influenza. [51] [55] Subtypes of IAV were discovered throughout the 1930s, [52] and IBV was discovered in 1940. [13]

During the Second World War, the US government worked on developing inactivated vaccines for influenza, resulting in the first influenza vaccine being licensed in 1945 in the United States. [5] ICV was discovered two years later in 1947. [13] In 1955, avian influenza was confirmed to be caused by IAV. [21] Four influenza pandemics have occurred since WWII, each less severe than the 1918 pandemic. The first of these was the Asian flu from 1957 to 1958, caused by an H2N2 strain [5] [35] and beginning in China's Yunnan province. The number of deaths probably exceeded one million, mostly among the very young and very old. [43] Notably, the 1957 pandemic was the first flu pandemic to occur in the presence of a global surveillance system and laboratories able to study the novel influenza virus. [20] After the pandemic, H2N2 was the IAV subtype responsible for seasonal influenza. [5] The first antiviral drug against influenza, amantadine, was approved for use in 1966, with additional antiviral drugs being used since the 1990s. [23]

In 1968, H3N2 was introduced into humans as a result of a reassortment between an avian H3N2 strain and an H2N2 strain that was circulating in humans. The novel H3N2 strain first emerged in Hong Kong and spread worldwide, causing the Hong Kong flu pandemic, which resulted in 500,000–2,000,000 deaths. This was the first pandemic to spread significantly by air travel. [19] [20] H2N2 and H3N2 co-circulated after the pandemic until 1971 when H2N2 waned in prevalence and was completely replaced by H3N2. [19] In 1977, H1N1 reemerged in humans, possibly after it was released from a freezer in a laboratory accident, and caused a pseudo-pandemic. [20] [52] Whether the 1977 "pandemic" deserves to be included in the natural history of flu pandemics is debatable. [43] This H1N1 strain was antigenically similar to the H1N1 strains that circulated prior to 1957. Since 1977, both H1N1 and H3N2 have circulated in humans as part of seasonal influenza. [5] In 1980, the current classification system used to subtype influenza viruses was introduced. [56]

At some point, IBV diverged into two lineages, named the B/Victoria-like and B/Yamagata-like lineages, both of which have been circulating in humans since 1983. [13] In 1996, HPAI H5N1 was detected in Guangdong, China [21] and a year later emerged in poultry in Hong Kong, gradually spreading worldwide from there. A small H5N1 outbreak in humans in Hong Kong occurred then, [28] and sporadic human cases have occurrence since 1997, carrying a high case fatality rate. [11] [37] The most recent flu pandemic was the 2009 swine flu pandemic, which originated in Mexico and resulted in hundreds of thousands of deaths. [20] It was caused by a novel H1N1 strain that was a reassortment of human, swine, and avian influenza viruses. [12] [23] The 2009 pandemic had the effect of replacing prior H1N1 strains in circulation with the novel strain but not any other influenza viruses. Consequently, H1N1, H3N2, and both IBV lineages have been in circulation in seasonal form since the 2009 pandemic. [5] [20] [21]

In 2011, IDV was discovered in pigs in Oklahoma, USA, and cattle were later identified as the primary reservoir of IDV. [7] [13] In the same year, [37] avian H7N9 was detected in China and began to cause human infections in 2013, starting in Shanghai and Anhui and remaining mostly in China. HPAI H7N9 emerged sometime in 2016 and has occasionally infected humans incidentally. Other AIVs have less commonly infected humans since the 1990s, including H5N6, H6N1, H7N2-4, H7N7, and H10N7-8, [11] and HPAI H subtypes such as H5N1-3, H5N5-6, and H5N8 have begun to spread throughout much of the world since the 2010s. Future flu pandemics, which may be caused by an influenza virus of avian origin, [21] are viewed as almost inevitable, and increased globalization has made it easier for novel viruses to spread, [20] so there are continual efforts to prepare for future pandemics [51] and improve the prevention and treatment of influenza. [5]

Etymology

The word influenza comes from the Italian word influenza, from medieval Latin influentia, originally meaning "visitation" or "influence" of the stars. This referred to the disease's cause, which at the time was ascribed by some to unfavorable astrological conditions. Its use in the disease sense is first attested in 1504, when it meant a "visitation" or "outbreak" of any disease affecting many people in a single place at once. During an outbreak of influenza in 1743 that started in Italy and spread throughout Europe, the word reached the English language and was anglicized in pronunciation. [57] [58] Other names that have been used for influenza include epidemic catarrh, la grippe from French, sweating sickness, and, especially when referring to the 1918 pandemic strain, Spanish fever. [59]

Influenza research is wide-ranging and includes efforts to understand how influenza viruses enter hosts, the relationship between influenza viruses and bacteria, how influenza symptoms progress, and what make some influenza viruses deadlier than others. [60] Non-structural proteins encoded by influenza viruses are periodically discovered and their functions are continually under research. [18] Past pandemics, and especially the 1918 pandemic, are the subject of much research to understand flu pandemics. [43] As part of pandemic preparedness, the Global Influenza Surveillance and Response System is a global network of laboratories that monitors influenza transmission and epidemiology. [61] Additional areas of research include ways to improve the diagnosis, treatment, and prevention of influenza.

Existing diagnostic methods have a variety of limitations coupled with their advantages. For example, NATs have high sensitivity and specificity but are impractical in under-resourced regions due to their high cost, complexity, maintenance, and training required. Low-cost, portable RIDTs can rapidly diagnose influenza but have highly variable sensitivity and are unable to subtype IAV. As a result of these limitations and others, research into new diagnostic methods revolves around producing new methods that are cost-effective, less labor-intensive, and less complex than existing methods while also being able to differentiate influenza species and IAV subtypes. One approach in development are lab-on-a-chips, which are diagnostic devices that make use of a variety of diagnostic tests, such as RT-PCR and serological assays, in microchip form. These chips have many potential advantages, including high reaction efficiency, low energy consumption, and low waste generation. [37]

New antiviral drugs are also in development due to the elimination of adamantines as viable drugs and concerns over oseltamivir resistance. These include: NA inhibitors that can be injected intravenously, such as intravenous formulations of zanamivir favipiravir, which is a polymerase inhibitor used against several RNA viruses pimodivir, which prevents cap-binding required during viral transcription and nitazoxanide, which inhibits HA maturation. [5] [8] Reducing excess inflammation in the respiratory tract is also subject to much research since this is one of the primary mechanisms of influenza pathology. [8] [9] Other forms of therapy in development include monoclonal and polyclonal antibodies that target viral proteins, convalescent plasma, different approaches to modify the host antiviral response, [29] [62] and stem cell-based therapies to repair lung damage. [8]

Much research on LAIVs focuses on identifying genome sequences that can be deleted to create harmless influenza viruses in vaccines that still confer immunity. [18] The high variability and rapid evolution of influenza virus antigens, however, is a major obstacle in developing effective vaccines. Furthermore, it is hard to predict which strains will be in circulation during the next flu season, manufacturing a sufficient quantity of flu vaccines for the next season is difficult, [14] LAIVs have limited efficacy, and repeated annual vaccination potentially has diminished efficacy. [5] For these reasons, "broadly-reactive" or "universal" flu vaccines are being researched that can provide protection against many or all influenza viruses. Approaches to develop such a vaccine include HA stalk-based methods such as chimeras that have the same stalk but different heads, HA head-based methods such as computationally optimized broadly neutralizing antigens, anti-idiotypic antibodies, and vaccines to elicit immune responses to highly conserved viral proteins. [14] [62] mRNA vaccines to provide protection against influenza are also under research. [63]

Birds

Aquatic birds such as ducks, geese, shorebirds, and gulls are the primary reservoir of IAVs. [11] [12] In birds, AIVs may be either low pathogenic avian influenza (LPAI) viruses that produce little to no symptoms or highly pathogenic avian influenza (HPAI) viruses that cause severe illness. Symptoms of HPAI infection include lack of energy and appetite, decreased egg production, soft-shelled or misshapen eggs, swelling of the head, comb, wattles, and hocks, purple discoloration of wattles, combs, and legs, nasal discharge, coughing, sneezing, incoordination, and diarrhea. Birds infected with an HPAI virus may also die suddenly without any signs of infection. [36]

The distinction between LPAI and HPAI can generally be made based on how lethal an AIV is to chickens. At the genetic level, an AIV can be usually be identified as an HPAI virus if it has a multibasic cleavage site in the HA protein, which contains additional residues in the HA gene. [12] [21] Most AIVs are LPAI. Notable HPAI viruses include HPAI H5N1 and HPAI H7N9. HPAI viruses have been a major disease burden in the 21st century, resulting in the death of large numbers of birds. In H7N9's case, some circulating strains were originally LPAI but became HPAI by acquiring the HA multibasic cleavage site. Avian H9N2 is also of concern because although it is LPAI, it is a common donor of genes to H5N1 and H7N9 during reassortment. [5]

Migratory birds can spread influenza across long distances. An example of this was when an H5N1 strain in 2005 infected birds at Qinghai Lake, China, which is a stopover and breeding site for many migratory birds, subsequently spreading the virus to more than 20 countries across Asia, Europe, and the Middle East. [11] [21] AIVs can be transmitted from wild birds to domestic free-range ducks and in turn to poultry through contaminated water, aerosols, and fomites. [5] Ducks therefore act as key intermediates between wild and domestic birds. [21] Transmission to poultry typically occurs in backyard farming and live animal markets where multiple species interact with each other. From there, AIVs can spread to poultry farms in the absence of adequate biosecurity. Among poultry, HPAI transmission occurs through aerosols and contaminated feces, [5] cages, feed, and dead animals. [11] Back-transmission of HPAI viruses from poultry to wild birds has occurred and is implicated in mass die-offs and intercontinental spread. [12]

AIVs have occasionally infected humans through aerosols, fomites, and contaminated water. [5] Direction transmission from wild birds is rare. [21] Instead, most transmission involves domestic poultry, mainly chickens, ducks, and geese but also a variety of other birds such as guinea fowl, partridge, pheasants, and quails. [12] The primary risk factor for infection with AIVs is exposure to birds in farms and live poultry markets. [11] Typically, infection with an AIV has an incubation period of 3–5 days but can be up to 9 days. H5N1 and H7N9 cause severe lower respiratory tract illness, whereas other AIVs such as H9N2 cause a more mild upper respiratory tract illness, commonly with conjunctivitis. [5] Limited transmission of avian H2, H5-7, H9, and H10 subtypes from one person to another through respiratory droplets, aerosols, and fomites has occurred, [5] [14] but sustained human-to-human transmission of AIVs has not occurred. Before 2013, H5N1 was the most common AIV to infect humans. Since then, H7N9 has been responsible for most human cases. [11]

Influenza in pigs is a respiratory disease similar to influenza in humans and is found worldwide. Asymptomatic infections are common. Symptoms typically appear 1–3 days after infection and include fever, lethargy, anorexia, weight loss, labored breathing, coughing, sneezing, and nasal discharge. In sows, pregnancy may be aborted. Complications include secondary infections and potentially fatal bronchopneumonia. Pigs become contagious within a day of infection and typically spread the virus for 7–10 days, which can spread rapidly within a herd. Pigs usually recover from infection within 3–7 days after symptoms appear. Prevention and control measures include inactivated vaccines and culling infected herds. The influenza viruses usually responsible for swine flu are IAV subtypes H1N1, H1N2, and H3N2. [64]

Some IAVs can be transmitted via aerosols from pigs to humans and vice versa. [5] Furthermore, pigs, along with bats and quails, [13] are recognized as a mixing vessel of influenza viruses because that have both α-2,3 and α-2,6 sialic acid receptors in their respiratory tract. Because of that, both avian and mammalian influenza viruses can infect pigs. If co-infection occurs, then reassortment is possible. [12] A notable example of this was the reassortment of a swine, avian, and human influenza virus in 2009, resulting in a novel H1N1 strain that caused the 2009 flu pandemic. [12] [23] Spillover events from humans to pigs, however, appear to be more common than from pigs to humans. [12]

Other animals

Influenza viruses have been found in many other animals, including cattle, horses, dogs, cats, and marine mammals. Nearly all IAVs are apparently descended from ancestral viruses in birds. The exception are bat influenza-like viruses, which have an uncertain origin. These bat viruses have HA and NA subtypes H17, H18, N10, and N11. H17N10 and H18N11 are unable to reassort with other IAVs, but they are still able to replicate in other mammals. [5] AIVs sometimes crossover into mammals. For example, in late 2016 to early 2017, an avian H7N2 strain was found to be infecting cats in New York. [5]

Equine IAVs include H7N7 and two lineages [5] of H3N8. H7N7, however, has not been detected in horses since the late 1970s, [16] so it may have become extinct in horses. [12] H3N8 in equines spreads via aerosols and causes respiratory illness. [5] Equine H3N8 perferentially binds to α-2,3 sialic acids, so horses are usually considered dead-end hosts, but transmission to dogs and camels has occurred, raising concerns that horses may be mixing vessels for reassortment. In canines, the only IAVs in circulation are equine-derived H3N8 and avian-derived H3N2. Canine H3N8 has not been observed to reassort with other subtypes. H3N2 has a much broader host range and can reassort with H1N1 and H5N1. An isolated case of H6N1 likely from a chicken was found infecting a dog, so other AIVs may emerge in canines. [12]

Other mammals to be infected by IAVs include H7N7 and H4N5 in seals, H1N3 in whales, and H10N4 and H3N2 in minks. [16] Various mutations have been identified that are associated with AIVs adapting to mammals. Since HA proteins vary in which sialic acids they bind to, mutations in the HA receptor binding site can allow AIVs to infect mammals. Other mutations include mutations affecting which sialic acids NA proteins cleave and a mutation in the PB2 polymerase subunit that improves tolerance of lower temperatures in mammalian respiratory tracts and enhances RNP assembly by stabilizing NP and PB2 binding. [12]

IBV is mainly found in humans but has also been detected in pigs, dogs, horses, and seals. [13] Likewise, ICV primarily infects humans but has been observed in pigs, dogs, cattle, and dromedary camels. [7] [13] IDV causes an influenza-like illness in pigs but its impact in its natural reservoir, cattle, is relatively unknown. It may cause respiratory disease resembling human influenza on its own, or it may be part of a bovine respiratory disease (BRD) complex with other pathogens during co-infection. BRD is a concern for the cattle industry, so IDV's possible involvement in BRD has led to research on vaccines for cattle that can provide protection against IDV. [13] [15] Two antigenic lineages are in circulation: D/swine/Oklahoma/1334/2011 (D/OK) and D/bovine/Oklahoma/660/2013 (D/660). [13]


Results

Decay of memory CD8 + T cells in the lung occurs with age

We measured the frequency of different immune cells present within human lung tissue from organ donors between the ages of 22� years. Lung sample preparations contained >ꁠ% haematopoietic cells (CD45 + ) and were banked prior to December 2019 (Figure  1a ). We observed no correlation in the frequency of B cells, MAIT cells, NK cells and CD4 + T cells and the age of the donor (Figure  1b and ​ andc), c ), although MAIT cells appear to follow a biphasic rise then fall with age, as previously reported. 6 , 7 Interestingly, the proportion of lung CD8 + T cells, the vast majority of which were antigen experienced (CD45RO + ), declined in the elderly, with these cells representing

ꀡ% of the total cells in lungs of donors <ꁐ years of age, but only

ਇ.5% of the total cells in donors >ꁐ years of age (Figure  1c and ​ andd). d ). Attrition of memory CD8 + T cells with age appears to be most pronounced in the lung environment, as assessment of the frequency of memory CD8 + T cells in the blood of a cohort of non‐matched donors showed that the proportion of memory CD8 + T cells does not wane with age (Supplementary figure 1), which is in line with our previous studies showing a gradual increase in the size of the circulating memory CD8 + T�ll pool with advanced age. 8 Assessment of the bulk CD8 + T�ll pool in the lung also revealed age‐related changes in T�ll subset composition, with naïve T cells (CD45RA + CD27 + ) declining and effector memory T cells (CD45RA − CD27 − ) increasing with advanced age (Supplementary figure 2). Interestingly, further phenotypic profiling of lung memory CD8 + T cells revealed that Trm, a memory T�ll subset, identified here by the co𠄎xpression of CD69 and the integrin CD103 9 also declined with age, with Trm representing

ꁈ% of total memory CD8 + T cells in donors <ꁐ years of age, but only

ꀥ% of the total lymphocytes in donors >ꁐ years of age (Figure  1e–g ). The decrease in the frequency of Trm coincided with a reciprocal increase in the proportion of CD69 − CD103 − memory CD8 + T cells (Figure  1f and ​ andg). g ). We have previously reported CD103 + CD69 + CD8 + Trm frequencies in another cohort of lung organ donors, 9 and when we combine the data acquired from both cohorts, thereby increasing the sample size to 14 donors, we still see a significant negative correlation between age and Trm frequency (Supplementary figure 3, Pearson r = 𢄠.608, *P =਀.021). Thus, the immune cell landscape in the human lung changes with advanced age, resulting in a decrease in the frequency of tissue‐residing memory CD8 + T cells.

Age𠄊ssociated decay of lung memory CD8 + T cells. (a) The proportion of CD45 + and CD45 − cells in the lung of donors. Bars represent individual donors, and symbols show the donor’s age. (b) Representative gating strategy for identifying immune cell subsets. (c) The frequency of B cells (CD19 + CD3 − ), CD3 + T cells (CD3 + CD19 − ), monocytes (CD3 − CD19 − CD56 − CD14 + ), γδ T cells (CD3 + CD161 − V㬗.2 − γδTCR + ), NK cells (CD3 − CD19 − CD56 + ), CD4 + T cells (CD3 + CD161 − γδTCR − CD4 + CD8 − ), MAIT cells (CD3 + CD161 + V㬗.2 + ) and CD8 + T cells (CD3 + CD161 − γδTCR − CD4 − CD8 + ) of total cells in the lungs plotted against age (years). (d) The frequency of CD45RO + CD8 + memory CD8 + T cells of cells in the lungs plotted against age (years). (e) Flow cytometry profiles depicting the level of expression of CD103 and CD69 on memory CD8 + T cells (CD45RO + CD8 + CD3 + ) isolated from human lung. (f) The frequency of CD8 + Trm (CD3 + CD45RO + CD8 + CD103 + CD69 + ) of total memory CD8 + T cells plotted against age (years). (g) The proportion of memory CD8 +  T cells (CD3 + CD45RO + CD8 + ) isolated from the lung that express CD103 and CD69. Bars represent individual donors. Symbols show the donor’s age (years). In c, d and f, symbols represent individual donors, and the line represents Pearson’s correlation. *P <਀.05 **P <਀.01.

Upregulation of activation markers on T cells in the lung following exposure to influenza virus decreases with age

We next investigated whether the age𠄊ssociated changes in the lung immune cell landscape impact the quality of the immune response mounted following exposure to influenza virus. To do this, we infected single�ll suspensions of whole lung tissues with influenza virus (H3N2, X31) at a multiplicity of infection (moi) of 1, and 24 h later measured the levels of expression markers indicative of immune cell activation, including CD38, HLA𠄍R, CD27, PD𠄁 and CD69, on both innate‐like (MAIT cells and γδ T cells) and conventional (CD4 and CD8) T cells. We limited our analysis here to the T�ll compartment as it was these cells that were most affected by donor age (see Figure  1 ). Influenza virus infection of human lung tissue caused the upregulation of expression of HLA𠄍R and CD69 on all T�ll subsets profiled, albeit with varying efficiencies, and upregulated expression of CD38 on both γδ T cells and CD8 + T cells (Figure  2a𠄾 ). Interestingly, when profiling the memory CD8 + T�ll pool, we observed that the level of upregulation of CD69 and HLA𠄍R negatively correlated with donor age (Figure  2e ). We checked the proportion of the lung cells that were infected with influenza virus, as assessed by intracellular staining for the viral nucleoprotein (NP), to rule out that the observed differences in T�ll activation across age groups was a consequence of variations in infection efficiency. We found that 1𠄵% of lung cells stained positive for influenza virus NP, and importantly, the level of infection was not influenced by the age of the donor (Supplementary figure 4). Thus, in addition to an age𠄊ssociated drop in the frequency of lung memory CD8 + T cells we also find that with advanced age, the bulk memory CD8 + T cells that are present in the lung undergo less activation following influenza virus exposure.

Upregulation of activation markers on innate and conventional T cells in the lung following exposure to influenza virus decreases with age. (a𠄾) Lung cells were infected with influenza virus (X31) at moi of 1, and expression of HLA𠄍R, CD27, CD38, CD69 and PD𠄁 on memory CD4 + T cells, memory CD8 + T cells, γδ T cells and MAIT cells was measured 24 h later. (a) Representative histograms show the expression level of activation markers post‐influenza virus exposure on specific immune cell subsets. Grey histograms show expression on cells from uninfected cultures. (b𠄾) Fold change in mean fluorescence intensity (MFI) of CD27, CD69, HLA𠄍R, CD38 and PD𠄁 on (b) MAIT cells, (c) γδ T cells, (d) memory CD4 + T cells and (e) memory CD8 + T cells present in influenza virus‐infected lung tissue relative to the expression on cells in uninfected control cultures (Nil) plotted against age (years). Symbols represent individual donors, and the line represents Pearson’s correlation. *P <਀.05.

Exposure of lung cells to influenza virus triggers an early pro‐inflammatory response that decreases with age

To determine whether the age𠄊ssociated changes in the immune cell landscape in the lung impact the inflammatory profile following exposure to influenza virus, single�ll suspensions of whole lung tissue were infected in vitro with mouse�pted influenza virus (H3N2, X31) at a moi of 1 and 24, and 48 h later, the level of a panel of cytokines in the supernatant was measured. Several pro‐inflammatory cytokines including TNF, IL𠄆, IFN‐㮱, IFN‐β, IL�, IL𠄈 and CXCL10 were induced following exposure to influenza virus, and the amount released appeared unaffected by the age of the donor (Figure  3a ). While influenza virus infection also caused the production of GM𠄌SF, IFNγ and IFNα, the amount of these cytokines produced at 24 and 48 h post‐infection negatively correlated with the age of the donor (Figure  3a ). Next, we tested whether infection with human influenza virus strains also triggered a similar inflammatory profile. To do this, single�ll suspensions of whole lung tissue were infected in vitro at a moi of 1 with either A/Sydney/203/2000 (H3N2) or A/Tasmania/2004/2009 (H1N1pdm09) and 24 and 48 h later, the level of infection, measured by intracellular NP staining, and the presence of GM𠄌SF, IFNγ and IFN㬒 in the supernatant was assessed. Similar to our earlier results, we did not observe any age𠄊ssociated impact on the ability of human influenza viruses to infect lung tissue, with 2.6�% of lung cells staining NP + following infection with A/Sydney/203/2000 and 1.8𠄶.2% of lung cells staining NP + following infection with A/Tasmania/2004/2009 (Supplementary figure 5a). In alignment with our observations following infection of human lung tissue with the mouse�pted X31 virus, we again observed that aged donors produce less IFN㬒, GM𠄌SF and IFNγ following infection with the human influenza isolates (Supplementary figure 5b𠄾). To gain insight into the cellular source of these cytokines, we repeated the experiment and this time added brefeldin A to the culture to trap cytokines intracellularly and profiled various immune cells including CD8 + T cells, CD4 + T cells, MAIT cells, NK cells and γδ T cells at 18 h post‐infection for the production of IFNγ and GM𠄌SF. Negligible levels of GM𠄌SF were detected in all immune cells profiled which suggests that another cell type not profiled in this assay is likely the source of this inflammatory cytokine (Figure  3b ). Assessment of IFNγ production revealed that memory CD8 + T cells were the main source and consistent with our earlier findings, the proportion of CD8 + memory T cells making IFNγ in response to influenza virus infection waned with age (Figure  3b and c ). Collectively, these results suggest that following infection with influenza virus, lung tissue from aged donors produces less IFNα, GM𠄌SF and IFNγ, the latter perhaps attributed by the reduction in IFNγ‐producing memory CD8 + T cells.

Exposure of lung cells to influenza virus triggers an early pro‐inflammatory response that decreases with age. (a) Lung cells were infected with influenza virus (X31) at moi of 1, and the levels of a panel of inflammatory cytokines released into the supernatant at 24 and 48 h were measured using a cytometric bead array. Graphs depict the amount (pg mL 𠄱 ) of inflammatory cytokine plotted against age (years). Symbols represent individual donors, and the dotted line represents the limit of detection (r = Pearson’s correlation). (b, c) Lung cells were infected with influenza virus (X31) at moi of 1 in the presence of brefeldin A, and the percentage of different immune cells generating intracellular IFNγ and GM𠄌SF was measured 18 h later. (b) Representative flow cytometry profile staining for GM𠄌SF and IFNγ on immune cell subsets with or without (Nil) virus infection. (c) The graph depicts the proportion of IFNγ‐producing memory CD8 + T cells with or without (Nil) virus infection plotted against age (years). Symbols represent individual donors, and the line represents Pearson’s correlation. *P <਀.05 **P <਀.01 ***P <਀.001.

Decay of influenza virus‐specific tissue‐resident memory T cell occurs with age

The diminished activation and IFNγ production by memory CD8 + T cells in the lungs of aged donors following exposure to influenza virus may be explained by a loss of influenza virus‐reactive memory CD8 + T cells in this tissue. To investigate this further, we measured the frequency of influenza‐specific memory CD8 + T cells in lung of donors using a panel of HLA‐peptide tetrameric complexes and enumerated the influenza virus‐specific cells by flow cytometry. Within our cohort, we identified 7 donors with HLA types for which HLA tetramers loaded with influenza immunodominant epitopes were available. These included donors who were HLA�, HLA� and HLA�. 10 The proportion of influenza‐specific (tetramer positive) CD8 + T cells of the total memory CD8 + T�ll pool across the donors ranged from 0.4% to 8.6%, and similar to our previously reported studies, 9 , 11 we observed no correlation between the size of the lung influenza‐specific memory CD8 + T�ll pool and donor age (Figure  4a and b ). These data suggest that the difference in memory CD8 + T�ll activation observed in aged donors following exposure to influenza virus is not simply because of a reduction in influenza virus‐specific CD8 + memory T cells. Interestingly, while the overall frequency of influenza‐specific lung memory CD8 + T cells was not affected by donor age, further inspection of this virus‐specific memory T�ll pool did reveal age‐related changes in memory T�ll subset composition. Consistent with our observed changes in the bulk memory CD8 + T�ll compartment, we found the proportion of CD103 + CD69 + influenza‐specific CD8 + Trm cells declined with age (Figure  4c ) and this coincided with a reciprocal increase in the proportion of CD69 − CD103 − memory CD8 + T cells (Figure  4d and e ). These results highlight the local resident memory CD8 + T�ll compartment in the lung declines with age, and this may, in part, impact the size and quality of the early inflammatory response evoked following virus exposure.

Decay of influenza virus‐specific CD8 + Trm occurs with age. (a) Representative flow cytometry staining assessing the expression of CD103 and CD69 on HLA�‐M158–specific CD8 + T cells isolated from lung tissue of two donors. (b) The percentages of influenza tetramer + CD8 + T cells of the total memory CD8 + T�ll pool (CD3 + CD8 + CD45RO + ) in the lungs of donors plotted against age (years). (c𠄾) The frequency of total memory CD8 + T cells and influenza tetramer + memory CD8 + T cells that are (c) CD103 + CD69 + Trm, (d) CD103 − CD69 − and (e) CD103 − CD69 + plotted against age (years). Symbols represent individual donors, and the line represents Pearson’s correlation. *P <਀.05 **P <਀.01 ***P <਀.001.

T cells in the lung fail to upregulate activation markers following exposure to SARS𠄌oV𠄂

We next investigated how the immune cells in the lung react following exposure to SARS𠄌oV𠄂, another respiratory pathogen which causes severe disease in the elderly. As all lung tissue was stored prior to the emergence of SARS𠄌oV𠄂, all donors will be immunologically naïve. Single�ll suspensions of whole lung tissue were infected with SARS𠄌oV𠄂 at a moi of 1, and 24 and 48 h later, the activation status of both innate‐like (or unconventional) and conventional T cells was measured by assessing the expression of HLA𠄍R, CD38, CD27, PD𠄁 and CD69. In parallel, as a positive control, we also infected cells with influenza virus. At 24 h post‐SARS𠄌oV𠄂 infection, irrespective of the donor age, we did not observe upregulation of any activation marker on any T�ll subset profiled (data not shown), a phenotype that was conserved at 48 h post‐SARS𠄌oV𠄂 infection (Figure  5a𠄽 ). In contrast, and consistent with our earlier 24‐h experiments, infection of the lung tissue with influenza virus for 48 h triggered both innate and conventional T�ll activation as measured by upregulation of HLA𠄍R and CD69 expression, and the upregulation of CD38 expression on both γδ T cells and CD8 + T cells (Figure  5a𠄽 ). The inability of SARS𠄌oV𠄂 infection to trigger immune cell activation was not because the virus was unable to infect these lung cells as assessment of viral RNA by RT‐qPCR at 1, 24 and 48 h post‐infection confirmed the presence of an increasing level of virus in all infected samples (Figure  5e–g ). Moreover, utilising a recombinant, fluorescently tagged SARS𠄌oV𠄂 RBD𠄍imer protein to identify angiotensin𠄌onverting enzyme 2 (ACE2), a transmembrane protein that serves as a receptor for entry of SARS𠄌oV𠄂 into host cells, we show that CD14 + cells in the human lungs can bind the RBD𠄍imer and the level of attachment does not appear to be influenced by donor age (Supplementary figure 6). To assess whether SARS𠄌oV𠄂 infection of the lung tissue triggers the release of pro‐inflammatory molecules, the levels of a panel of cytokines released into the supernatants were measured. Of interest, while influenza virus infection triggered the production of type I, II and III interferon, infection with SARS𠄌oV𠄂 did not evoke production of any of these interferons (Figure  6 ). Collectively, these results show that irrespective of donor age, exposure of human lung cells to SARS𠄌oV𠄂 does not trigger the activation of local immune cells and does not result in the induction of an early interferon response.

Innate‐like and conventional T cells in the lung fail to upregulate activation markers following exposure to SARS𠄌oV𠄂. (a𠄽) Cells from whole lung tissue were infected with either influenza virus (X31) or SARS𠄌oV𠄂 at moi of 1, and expression of HLA𠄍R, CD69, CD38, CD27 and PD𠄁 on MAIT cells, memory CD4 + T cells, memory CD8 + T cells and γδ T cells was measured 48 h later. Fold change in MFI of HLA𠄍R, CD69, CD38, CD27 and PD𠄁 on (a) memory CD8 + T cells (b) memory CD4 + T cells (c) γδ T cells and (d) MAIT cells in virus‐infected lung tissue relative to expression on cells from uninfected control cultures is plotted against age (years). Symbols represent individual donors, and the line represents Pearson’s correlation. The dotted line at 1 represents no change in marker expression post‐infection. (e–g) Cells from whole lung tissue were infected with SARS𠄌oV𠄂 at a moi of 1 or mock infected, and at 1, 24 and 48 h later, the amount of viral RNA was measured by RT‐qPCR. Each graph depicts the mean (two technical replicates), pooled from two experiments amount of viral RNA (ng μL 𠄱 ) per donor plotted against (e) time or (f, g) age (years). *P <਀.05.

Infection of lung cells with SARS𠄌oV𠄂 fails to evoke an early interferon response. Cells from lung tissue were infected with either influenza virus (X31) or SARS𠄌oV𠄂 at moi of 1. and the levels of IFNα, IFNγ and IFN㮱 released into the supernatant at 24 and 48 h were measured using a cytometric bead array. Graphs depict the amount of inflammatory cytokine. Symbols represent individual donors (two‐way ANOVA, Sidak’s multiple comparison). The dotted line is the limit of detection at 2 pg mL 𠄱 . *P <਀.05 ****P < 0.0001.


Influenza Virus (Flu)

Nearly everyone has experienced the fever, aches, and other symptoms of seasonal flu that afflicts 5 – 20 percent of Americans each year. Although these yearly flu epidemics can be fatal in some people, such as the elderly, young children, and people with certain underlying heath conditions, flu is generally not a life-threatening disease in healthy individuals.

Flu, or influenza, is a contagious respiratory illness that spreads from person to person through the air via coughs or sneezes or through contact with infected surfaces. It is caused by a group of continuously changing viruses called influenza viruses.

Influenza viruses change easily and often, they are unpredictable, and they can be deadly. It is always a great concern when a new flu virus emerges, because the general population does not have immunity and almost everyone is susceptible to infection and disease.

Every few decades or so, a new version of the influenza virus emerges in the human population that causes a serious global outbreak of disease called a pandemic. Pandemics are associated with widespread illness - and sometimes death - even in otherwise healthy people. These outbreaks can also lead to social disruption and economic loss.

About a decade ago, scientists and public health officials feared that we might be on the brink of a pandemic caused by the so-called avian or bird H5N1 flu that began circulating among poultry, ducks, and geese in Asia and spread to Europe and Africa. To date, the avian flu virus has not acquired to ability to spread easily from person to person – a necessary step in order for a virus to cause a pandemic.

In the spring of 2009, a different influenza virus - one that had never been seen before - suddenly appeared. The novel virus, commonly called swine flu, is named influenza A (H1N1). Unlike the avian H5N1 flu, the H1N1 swine flu is capable of being transmitted easily from person to person. Fortunately, however, H1N1 is far less deadly than the H5N1 virus. In only a few short weeks after emerging in North America, the new H1N1 virus reached around the world. As a result of the rapid, global spread of H1N1, the first pandemic of the 21st century was declared in June of 2009.

Although the 2009 H1N1 pandemic did not turn out to be as deadly as initially feared, the next pandemic flu virus could emerge at any time, and we must remain vigilant. Hopefully, the knowledge gained in response to the H5N1 and 2009 H1N1 outbreaks, and continued research to more completely understand influenza virus, as well as improvements in vaccine and drug development, will enable us to minimize the effects of future influenza outbreaks.

Different Types of Influenza Virus

There are three different types of influenza virus – A, B, and C. Type A viruses infect humans and several types of animals, including birds, pigs, and horses. Type B influenza is normally found only in humans, and type C is mostly found in humans, but has also been found in pigs and dogs.

Influenza pandemics are caused by type A viruses, and therefore these are the most feared type of influenza virus neither types B or C have caused pandemics.

Type A influenza is classified into subtypes depending on which versions of two different proteins are present on the surface of the virus. These proteins are called hemagglutinin (HA) and neuraminidase (NA). There are 17 different versions of HA and 10 different versions of NA. So, for example, a virus with version 1 of the HA protein and version 2 of the NA protein would be called influenza A subtype H1N2 (A H1N2, for short).

The influenza A subtypes are further classified into strains, and the names of the virus strains include the place where the strain was first found and the year of discovery. Therefore, an H1N1 strain isolated in California in 2009 is referred to as A/California/07/2009 (H1N1).

Although many different combinations of the HA and NA proteins are possible, viruses with only a few of the possible combinations circulate through the human population at any given time. Currently, subtypes H1N1 and H3N2 are in general circulation in people. Other combinations circulate in animals, such as the H5N1 virus found in birds. The subtypes that exist within a population change over time. For example, the H2N2 subtype, which infected people between 1957 and 1968, is no longer found in humans.

What Influenza Viruses Are Made of

Influenza virus has a rounded shape (although it can be elongated or irregularly shaped) and has a layer of spikes on the outside.

There are two different kinds of spikes, each made of a different protein – one is the hemagglutinin (HA) protein and the other is the neuraminidase (NA) protein.

The HA protein allows the virus to stick to a cell, so that it can enter into a host cell and start the infection process (all viruses need to enter cells in order to make more copies of themselves).

The NA protein is needed for the virus to exit the host cell, so that the new viruses that were made inside the host cell can go on to infect more cells. Because these proteins are present on the surface of the virus, they are “visible” to the human immune system.

Inside the layer of spikes, there are eight pieces, or segments, of RNA that contain the genetic information for making new copies of the virus. Each of these segments contains the instructions to make one or more proteins of the virus. So for example, segment 4 contains the instructions to make the HA protein, and segment 6 contains the instructions to make the NA protein (the segments are numbered in size order, with 1 being the largest).

When new viruses are made inside the host cell, all eight segments need to be assembled into a new virus particle, so that each virus has the complete set of instructions for making a new virus. The danger occurs when there are two different subtypes of influenza A inside the same cell, and the segments become mixed to create a new virus.

How Influenza Viruses Change

Influenza virus is one of the most changeable viruses known. There are two ways that influenza virus changes – these are called drift and shift.

Drifting, or antigenic drift, is a gradual, continuous change that occurs when the virus makes small “mistakes” when copying its genetic information. This can result in a slight difference in the HA or NA proteins. Although the changes may be small, they may be significant enough so that the human immune system will no longer recognize and defend against the altered proteins. This is why you can repeatedly get the flu and why flu vaccines must be administered each year to combat the current circulating strains of the virus.

Shifting, or antigenic shift, is an abrupt, major change in the virus, which produces a new combination of the HA and NA proteins. These new influenza virus subtypes have not been seen in humans (or at least not for a very long time), and because they are so different from existing influenza viruses, people have very little protection against them. When this happens, and the newly created subtype can be transmitted easily from one person to another, a pandemic could occur.

Virus shift can take place when a person or animal is infected with two different subtypes of influenza. Take the case, for example, where there are two different subtypes of influenza circulating at the same time, one in humans and one in ducks. The human subtype is able to infect humans and pigs, but not ducks, while the duck subtype is able to infect ducks and pigs, but not humans. When a pig becomes infected with both the human and duck influenza subtypes at the same time, the segments of both viruses are scrambled or reassorted. inside an infected pig cell. As a result, a human virus particle could assemble that contains the duck HA segment instead of the human HA segment. A new virus subtype has been created. This new subtype could infect humans, but because it has the new duck version of the HA protein, the human immune system would not be able to defend an infected person against the new virus subtype. The virus could continue to change to allow it to spread more easily in its new host, and widespread illness and death could result.

Virus shift can also occur when an avian strain becomes adapted to humans, so that the avian virus is easily transmitted from person to person. In this case, the avian strain jumps directly from birds to humans, without mixing or reassortment of the genetic material of influenza strains from different species.

Influenza Epidemics and Pandemics

Influenza epidemics, also known as seasonal flu, occur annually and are the most common emerging infection among humans. These epidemics have major medical impacts, but they are generally not fatal except in certain groups such as the elderly.

Pandemics, on the other hand, happen once every few decades on average. They occur when a new subtype of influenza A arises that has either never circulated in the human population or has not circulated for a very long time (so that most people do not have immunity against the virus). The new subtype often causes serious illness and death, even among healthy individuals, and can spread easily through the human population.

There were three influenza pandemics in the 20th century – the “Spanish” flu of 1918-19, the “Asian” flu of 1957-58, and the “Hong Kong” flu of 1968-69. The 1918 flu, caused by a strain of H1N1, was by far the most deadly. More than 500,000 people died in the United States as a result of the Spanish flu, and up to 50 million people may have died worldwide. Nearly half of those of those deaths were among young, otherwise healthy individuals. The 1957 pandemic was due to a new H2N2 strain of influenza virus that caused the deaths of two million people, while the 1968 pandemic resulted from an H3N2 strain that killed one million people.

One pandemic has occurred so far in the 21st century. This was due to the novel swine-origin H1N1 virus which emerged in 2009.

The WHO established a six phase pandemic alert system in 2005 in response to the potential threat of the H5N1 avian influenza virus. The alert system is based on the geographic spread of the virus, not necessarily the severity of disease caused by the virus. Although a disease may be “moderate” in severity, during widespread outbreaks, declaration of a pandemic is beneficial because it accelerates the vaccine production and prompts governments to take extra measures to contain the virus. Travel and trade bans may be implemented in some cases, although if the disease is already widespread, these may not be considered effective.

Prior to the emergence of the 2009 H1N1 virus, the alert level stood at Phase 3 based on the circulation of the H5N1 virus. On April 27, 2009, after the H1N1 flu virus was recognized to be passing from person to person in Mexico, the alert level was raised to Phase 4. Two days later, on April 29, the WHO again increased the alert level, this time to Phase 5, reflecting the sustained transmission of the novel H1N1 virus in the United States. As H1N1 continued to spread worldwide and infect people in over 70 countries, the WHO raised the alert to Phase 6 – the highest level - on June 11, 2009. Over the next few months, H1N1 spread to more than 200 countries and territories worldwide. The Phase 6 alert of the 2009 H1N1 pandemic was declared by the WHO to have ended on Aug. 10, 2010.

Avian Flu

Influenza naturally infects wild birds all around the world, although they usually do not become ill. The virus is very contagious, however, and it can become a problem when the virus is transmitted to domesticated birds, such as chickens, ducks, or turkeys, because domesticated poultry can succumb to illness and death from influenza.

Humans generally do not become infected with avian flu. That is why news of humans contracting avian influenza during an outbreak of bird flu in poultry in 1997 in Hong Kong was alarming. It indicated that the virus had changed to allow it to directly infect humans. The virus that caused this particular outbreak is influenza A subtype H5N1.

Since 1997, H5N1 infections in birds have spread, initially throughout Asia. Then as birds traveled along their migratory routes, H5N1 dispersed to Russia and Europe, and later to countries in the Middle East and on the African continent.

Most human cases of H5N1 influenza have been traced to direct contact with infected poultry, but there have been a few cases of person-to-person transmission, particularly in clusters where multiple family members became infected.

One reason why avian H5N1 is not readily transmissible among people has to do with the hemagglutinin, or HA, protein of the virus that determines which cell type the virus can enter. As with other viruses, the influenza virus must attach to specific proteins called receptors on the outside of cells in order to gain entry into cells and cause an infection. Unlike human influenza viruses, which infect cells high in the respiratory tract, the H5N1 HA protein attaches to cells much lower in the respiratory track. The virus is so deep within the respiratory tract that it is not coughed up or sneezed out, and so it does not easily infect other people. If the HA protein of H5N1 were to mutate so that it could infect cells higher in the respiratory tract, then it would more likely be able to pass from person to person.

As of July 2015, there have been some 840 laboratory-confirmed cases of H5N1 infections in humans, in 16 different countries, and close to 450 deaths. The countries with the overall highest case numbers are Egypt, where almost all cases in 2015 have occurred, followed by Indonesia and Vietnam.

H5N1 continues to circulate in poultry, and small and sporadic clusters of human infections are still occurring. However, H5N1 currently does not transmit easily between people, so the risk of a large outbreak is low at this time.

Highly pathogenic H5 avian virus infections were first reported in birds in the United States in December 2014. Over approximately the next six months, more than 200 findings of infection with H5N2, H5N8, and H5N1 viruses were confirmed, mostly in poultry including backyard and commercial flocks. More than 40 million birds in 20 states were either infected or exposed. No human infections by these H5 viruses have been reported in the United States, but their presence in birds makes it more likely than human H5 infections could occur in the United States. Individuals having close contact with live infected poultry or surfaces contaminated with the avian influenza viruses are at highest risk of infection in places where the viruses circulate. There have been no reports of infection occurring from eating properly cooked poultry.

In addition to the H5 viral subtypes, other avian influenza strains have occasionally infected humans in recent years. These include the H7N2 strain which infected two individuals in the eastern United States in 2002 and 2003, and the H9N2 strain which has caused illness in several people in Asia in 1999 and 2003.

In March of 2013, a new subtype of avian influenza was found to infect humans. Influenza A (H7N9) had previously been detected in birds, but this particular variant had never been seen before in humans or animals. The initial wave of H7N9 infections occurred in the spring of 2013 in China, followed by a larger, second wave in the first half of 2014 in China and a few neighboring countries. As of February 2015, approximately 570 cases and 210 deaths have been reported to the WHO, mostly in China. People in the majority of cases had exposure to infected poultry or contaminated environments. The H7N9 virus causes a severe respiratory illness in most infected people, but it currently does not appear to spread easily from person to person.

Swine Flu

Swine influenza, or swine flu, is a very contagious respiratory disease of pigs. Although pigs become ill, they generally do not die from swine flu viruses.

In April of 2009, an influenza virus originating in swine was discovered to be capable of infecting humans and spreading from person to person. The new virus is named influenza A (H1N1), although it is commonly referred to as swine flu. Although it is called swine flu, the new H1N1 virus is transmitted from person to person, and not through contact with pigs or pork products.

The new H1N1 virus is made up of a novel combination of segments from four different influenza virus strains - a Eurasian swine virus, a North American swine virus, and avian and human influenza virus segments. Reassortment of segments from these different viruses produced a unique virus that had not been seen before by the human population. When novel viruses like this emerge, natural immunity is usually limited or nonexistent in humans.

The H1N1 influenza virus outbreak originated in Mexico in early 2009, and then spread rapidly throughout North America. Within a few weeks, the novel swine-origin H1N1 virus extended its reach around the globe. In June 2009, as a result of the global spread of the H1N1 virus, the WHO issued its first pandemic declaration of the 21st century - the first since the flu pandemic of 1968. The pandemic declaration acknowledged the inability to contain the virus and recognized its inevitable further spread within affected countries and into new countries. The new H1N1 virus became the dominant influenza strain in most parts of the world, including the United States.

Like other influenza pandemics, the 2009 H1N1 outbreak occurred in waves. The first wave took place in the spring of 2009, with a second wave commencing in late August as children and college students returned to classes. The outbreak peaked in October of 2009, with flu activity reported in all 50 states, as well as numerous other countries and territories. By January 2010, flu activity had returned to below baseline levels.

The H1N1 virus continues to circulate at low levels, but it is no longer the dominant influenza strain, and its behavior more closely resembles a seasonal influenza virus than a pandemic flu.

From the time the outbreak began in April 2009 through April 2010, the CDC estimated that about 60 million Americans became infected with the H1N1 virus, 265,000 Americans were hospitalized and 12,000 deaths occurred as a consequence of the 2009 H1N1 flu. The highest hospitalization rates occurred in young children. Exact numbers are not known due to the widespread nature of the outbreak and because most patients, especially those with mild cases, were not tested. The large majority of infections in the United States and most other countries were mild, although pregnant women and individuals with certain underlying medical conditions had an increased risk of severe and fatal illness.

There were some differences between the pandemic H1N1 flu and regular, seasonal flu. First, the H1N1 flu continued to spread during the summer months, which is uncommon for seasonal flu. Second, a much larger percentage of H1N1 patients exhibited symptoms of vomiting and diarrhea than is common with regular seasonal flu. There were also more reports of severe respiratory disease, especially in young and otherwise healthy people, infected with the new H1N1 virus than with seasonal flu viruses.

Significantly, the majority of cases of H1N1 infection, including severe and fatal cases, occurred in young and otherwise healthy individuals generally between the ages of 5 and 50, with relatively few deaths among the elderly. This is in contrast to the situation with seasonal flu which primarily afflicts the very young and the elderly, and where 90 percent of severe and lethal cases occur in people over the age of 65. Deaths among the elderly accounted for only 11 percent of H1N1 deaths.

Fortunately, the 2009 H1N1 flu was sensitive to two antiviral drugs used to treat influenza - Tamiflu® (oseltamivir) and Relenza® (zanamivir). The drugs act by inhibiting the essential neuraminidase protein (the “N” protein in the naming system). Proper use of these drugs can shorten the duration and lessen the severity of the sickness and reduce the chance of spreading the disease. The drugs reduce the risk of pneumonia - a major cause of death from influenza - and the need for hospitalization. To be most effective, the antiviral drugs should be administered as soon as possible after the onset of symptoms.

A vaccine to protect against the H1N1 virus was developed, tested, and approved and became available in October 2009. Due to the fact that the virus used to prepare the vaccine grew more slowly than most seasonal flu viruses do, production of the vaccine lagged and widespread distribution of the vaccine occurred later than anticipated. Priority for the vaccine was initially given to health care and emergency workers and individuals at high risk for severe disease, but by the winter of 2009-2010 availability was extended to the general population. Later, some doses went unused.

Although some had concerns about the safety of the H1N1 vaccine, flu vaccines have a very good safety profile. While mild side effects, such as soreness at the site of injection, aches, and low-grade fever, may occur as a result of receiving a flu shot, it is not possible to get the flu (H1N1 or seasonal) from the vaccine. The flu shot, or inactivated vaccine, is made from only a portion of the virus – a purified protein that makes our immune system develop protection. Likewise, the nasal spray version of the flu vaccine contains attenuated or weakened virus that is not able to cause the flu. Given the potential serious health outcomes from the flu, especially for high-risk population groups, the benefits of vaccination as the best way to prevent influenza infection and its complications far outweigh the risk of relatively minor side effects from the vaccination.


Contents

In 1930, two major categories of H. influenzae were defined: the unencapsulated strains and the encapsulated strains. Encapsulated strains were classified on the basis of their distinct capsular antigens. The six generally recognized types of encapsulated H. influenzae are: a, b, c, d, e, and f. [6] Genetic diversity among unencapsulated strains is greater than within the encapsulated group. Unencapsulated strains are termed nontypable (NTHi) because they lack capsular serotypes however, they can be classified by multilocus sequence typing. The pathogenesis of H. influenzae infections is not completely understood, although the presence of the polyribosyl ribitol phosphate (PRP) capsule in encapsulated type b (Hib), a serotype causing conditions such as epiglottitis, is known to be a major factor in virulence. [7] Their capsule allows them to resist phagocytosis and complement-mediated lysis in the nonimmune host. The unencapsulated strains are almost always less invasive they can, however, produce an inflammatory response in humans, which can lead to many symptoms. Vaccination with Hib conjugate vaccine is effective in preventing Hib infection but does not prevent infection with NTHi strains. [8]

Most strains of H. influenzae are opportunistic pathogens that is, they usually live in their host without causing disease, but cause problems only when other factors (such as a viral infection, reduced immune function or chronically inflamed tissues, e.g. from allergies) create an opportunity. They infect the host by sticking to the host cell using trimeric autotransporter adhesins. [ citation needed ]

Haemophilus influenzae type b (Hib) infection Edit

Naturally acquired disease caused by H. influenzae seems to occur in humans only. In infants and young children, H. influenzae type b (Hib) causes bacteremia, pneumonia, epiglottitis and acute bacterial meningitis. On occasion, it causes cellulitis, osteomyelitis, and infectious arthritis. It is one cause of neonatal infection. [9]

Due to routine use of the Hib vaccine in the U.S. since 1990, the incidence of invasive Hib disease has decreased to 1.3/100,000 in children. However, Hib remains a major cause of lower respiratory tract infections in infants and children in developing countries where the vaccine is not widely used. Unencapsulated H. influenzae strains are unaffected by the Hib vaccine and cause ear infections (otitis media), eye infections (conjunctivitis), and sinusitis in children, and are associated with pneumonia. [ citation needed ]

Clinical features may include initial symptoms of an upper respiratory tract infection mimicking a viral infection, usually associated with fevers, often low-grade. This may progress to the lower respiratory tract in a few days, with features often resembling those of a wheezy bronchitis. Sputum may be difficult to expectorate and is often grey or creamy in color. The cough may persist for weeks without appropriate treatment. Many cases are diagnosed after presenting chest infections that do not respond to penicillins or first-generation cephalosporins. A chest X-ray can identify alveolar consolidation. [10]

Clinical diagnosis of H. influenzae is typically performed by bacterial culture or latex particle agglutinations. Diagnosis is considered confirmed when the organism is isolated from a sterile body site. In this respect, H. influenzae cultured from the nasopharyngeal cavity or sputum would not indicate H. influenzae disease, because these sites are colonized in disease-free individuals. [11] However, H. influenzae isolated from cerebrospinal fluid or blood would indicate H. influenzae infection.

Culture Edit

Bacterial culture of H. influenzae is performed on agar plates, the preferable one being chocolate agar, with added X (hemin) and V (nicotinamide adenine dinucleotide) factors at 37 °C in a CO2-enriched incubator. [12] Blood agar growth is only achieved as a satellite phenomenon around other bacteria. Colonies of H. influenzae appear as convex, smooth, pale, grey, or transparent colonies. [ citation needed ]

Gram stained and microscopic observation of a specimen of H. influenzae will show Gram-negative coccobacillus. The cultured organism can be further characterized using catalase and oxidase tests, both of which should be positive. Further serological testing is necessary to distinguish the capsular polysaccharide and differentiate between H. influenzae b and nonencapsulated species. [ citation needed ]

Although highly specific, bacterial culture of H. influenzae lacks sensitivity. Use of antibiotics prior to sample collection greatly reduces the isolation rate by killing the bacteria before identification is possible. [13] Beyond this, H. influenzae is a finicky bacterium to culture, and any modification of culture procedures can greatly reduce isolation rates. Poor quality of laboratories in developing countries has resulted in poor isolation rates of H. influenzae. [ citation needed ]

H. influenzae will grow in the hemolytic zone of Staphylococcus aureus on blood agar plates the hemolysis of cells by S. aureus releases factor V which is needed for its growth. H. influenzae will not grow outside the hemolytic zone of S. aureus due to the lack of nutrients such as factor V in these areas. Fildes agar is best for isolation. In Levinthal medium, capsulated strains show distinctive iridescence.

Latex particle agglutination Edit

The latex particle agglutination test (LAT) is a more sensitive method to detect H. influenzae than is culture. [14] Because the method relies on antigen rather than viable bacteria, the results are not disrupted by prior antibiotic use. It also has the added benefit of being much quicker than culture methods. However, antibiotic sensitivity testing is not possible with LAT alone, so a parallel culture is necessary. [ citation needed ]

Molecular methods Edit

Polymerase chain reaction (PCR) assays have been proven to be more sensitive than either LAT or culture tests, and highly specific. [14] However, PCR assays have not yet become routine in clinical settings. Countercurrent immunoelectrophoresis has been shown to be an effective research diagnostic method, but has been largely supplanted by PCR.

Both H. influenzae and S. pneumoniae can be found in the upper respiratory system of humans. In an in vitro study of competition, S. pneumoniae always overpowered H. influenzae by attacking it with hydrogen peroxide and stripping off the surface molecules H. influenzae needs for survival. [15]

When both bacteria are placed together into a nasal cavity, within 2 weeks, only H. influenzae survives. When either is placed separately into a nasal cavity, each one survives. Upon examining the upper respiratory tissue from mice exposed to both bacteria species, an extraordinarily large number of neutrophils (immune cells) was found. In mice exposed to only one of the species, the neutrophils were not present.

Lab tests showed neutrophils exposed to dead H. influenzae were more aggressive in attacking S. pneumoniae than unexposed neutrophils. Exposure to dead H. influenzae had no effect on live H. influenzae.

Two scenarios may be responsible for this response:

  1. When H. influenzae is attacked by S. pneumoniae, it signals the immune system to attack the S. pneumoniae
  2. The combination of the two species triggers an immune system response that is not set off by either species individually.

It is unclear why H. influenzae is not affected by the immune response. [16]

Haemophilus influenzae can cause respiratory tract infections including pneumonia, otitis media, epiglottitis (swelling in the throat), eye infections and bloodstream infection, meningitis. It can also cause cellulitis (skin infection) and infectious arthritis (inflammation of the joint). [17]

Haemophilus influenzae produces beta-lactamases, and it is also able to modify its penicillin-binding proteins, so it has gained resistance to the penicillin family of antibiotics. In severe cases, cefotaxime and ceftriaxone delivered directly into the bloodstream are the elected antibiotics, and, for the less severe cases, an association of ampicillin and sulbactam, cephalosporins of the second and third generation, or fluoroquinolones are preferred. (Fluoroquinolone-resistant Haemophilus influenzae have been observed.) [18]

Macrolides and fluoroquinolones have activity against non-typeable H. influenzae and could be used in patients with a history of allergy to beta-lactam antibiotics. [19] However, macrolide resistance has also been observed. [20]

The serious complications of HiB are brain damage, hearing loss, and even death. [21]

Effective vaccines for Haemophilus influenzae Type B have been available since the early 1990s, and are recommended for children under age 5 and asplenic patients. The World Health Organization recommends a pentavalent vaccine, combining vaccines against diphtheria, tetanus, pertussis, hepatitis B and Hib. There is not yet sufficient evidence on how effective this pentavalent vaccine is in relation to the individual vaccines. [22]

Hib vaccines cost about seven times the total cost of vaccines against measles, polio, tuberculosis, diphtheria, tetanus, and pertussis. Consequently, whereas 92% of the populations of developed countries were vaccinated against Hib as of 2003, vaccination coverage was 42% for developing countries, and only 8% for least-developed countries. [23]

The Hib vaccines do not provide cross-protection to any other Haemophilus influenzae serotypes like Hia, Hic, Hid, Hie or Hif. [24]

An oral vaccination has been developed for non-typeable Haemophilus influenzae (NTHi) for patients with chronic bronchitis but it has not shown to be effective in reducing the number and severity of COPD exacerbations. [25]

H. influenzae was the first free-living organism to have its entire genome sequenced. Completed by Craig Venter and his team at The Institute for Genomic Research, one of the institutes now part of the J. Craig Venter Institute. Haemophilus was chosen because one of the project leaders, Nobel laureate Hamilton Smith, had been working on it for decades and was able to provide high-quality DNA libraries. The genome of strain Rd KW20 consists of 1,830,138 base pairs of DNA in a single circular chromosome that contains 1604 protein-coding genes, 117 pseudogenes, 57 tRNA genes, and 23 other RNA genes. [26] The sequencing method used was whole-genome shotgun, which was completed and published in Science in 1995. [27]

Unencapsulated H. influenzae is often observed in the airways of patients with chronic obstructive pulmonary disease (COPD). Neutrophils are also observed in large numbers in sputum from patients with COPD. The neutrophils phagocytize H. influenzae, thereby activating an oxidative respiratory burst. [28] However instead of killing the bacteria the neutrophils are themselves killed (though such an oxidative burst likely causes DNA damage in the H. influenzae cells). Dearth of killing the bacteria appears to explain the persistence of infection in COPD. [28]

H. influenzae mutants defective in the rec1 gene (a homolog of recA) are very sensitive to killing by the oxidizing agent hydrogen peroxide. [29] This finding suggests that rec1 expression is important for H. influenzae survival under conditions of oxidative stress. Since it is a homolog of recA, rec1 likely plays a key role in recombinational repair of DNA damage. Thus H. influenzae may protect its genome against the reactive oxygen species produced by the host's phagocytic cells through recombinational repair of oxidative DNA damages. [30] Recombinational repair of a damaged site of a chromosome requires, in addition to rec1, a second homologous undamaged DNA molecule. Individual H. influenzae cells are capable of taking up homologous DNA from other cells by the process of transformation. Transformation in H. influenzae involves at least 15 gene products, [27] and is likely an adaptation for repairing DNA damage in the resident chromosome. [ citation needed ]

Vaccines that target unencapsulated H. influenzae serotypes are in development. [31]


Biology of emerging viruses SARS, avian and human influenza, metapneumovirus, Nipah virus, West Nile, and the Ross River virus.

Biology of emerging viruses SARS, avian and human influenza, metapneumovirus, Nipah virus, West Nile, and the Ross River virus.

Annals of the New York Academy of Sciences v.1102

They have come and gone since the beginning of time and few people knew or cared much about them. Now we know more about what they can do, the devastation they can cause, and the cost in human life and well-being. This collection from the April 2005 meeting titled "Emerging Viral Infectious Diseases" in Hanoi details the viruses causing the most concern and acknowledges the role of globalization in the initiation and spread of viruses to which humans are vulnerable. They note that whole economies, particularly those of developing countries, can collapse or be irretrievably impaired by the onset of a pandemic or suspicion that one may be coming. Each of the papers focuses on a different strain, and includes recent findings about the virus at the molecular level, indicators of progress and current efforts to medically eradicate or control the virus in question.


Associated symptoms

Characteristic influenza symptoms are very high fever associated with respiratory issues such as dry “goose-honking” cough and labored abdominal breathing also called thumping.

Apathy and anorexia are also typical of this disease. When one walks into a barn affected by swine influenza, the pigs are laying down, there is no movement, it is very difficult to get them to move. Conjunctivitis and nasal discharge may also be seen. Symptoms usually last about a week and can range widely in severity from pig to pig. Mortality is close to 100% if there is no immunity but mortality is rare if the disease is not complicated by co-infections. Outbreaks in the breeding herd lead to reproductive issues such as increased stillbirths, abortions, and lowered fertility. This cannot be linked directly to the virus, as the virus does not replicate in any part of the reproductive system but is a consequence of the high fever associated with the infection.

Swine influenza can be transmitted from the dam to the piglets in utero. True or False?


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Keywords: COVID-19, Markov, probability, symptoms, stochastic, model, disease, influenza

Citation: Larsen JR, Martin MR, Martin JD, Kuhn P and Hicks JB (2020) Modeling the Onset of Symptoms of COVID-19. Front. Public Health 8:473. doi: 10.3389/fpubh.2020.00473

Received: 13 April 2020 Accepted: 27 July 2020
Published: 13 August 2020.

Alexander Rodriguez-Palacios, Department of Medicine, Case Western Reserve University, United States

Herve Seligmann, The Hebrew University of Jerusalem, Israel
Tauqeer Hussain Mallhi, Department of Clinical Pharmacy, College of Pharmacy, Al Jouf University, Saudi Arabia

Copyright © 2020 Larsen, Martin, Martin, Kuhn and Hicks. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.