Table of contents :

Epidemiology : first described by Hippocrates in 412 b.C.. Different strains also infect Aves spp. (chickens, turkeys, ostriches (various AI virus strains have been isolated in recent years from clinically affected ostriches, in several countries. All but one were not poultry-pathogenic : the only reported clinical outbreak in ostriches caused by a poultry-pathogenic strain (HPAI) was recorded in Italy (H₇N₁)^ref), quail, and peacocks; aquatic species : ducks, geese), Sus scrofa, Equus caballus, Phocidae, mustelids and Bos taurus^ref. Although viruses of relatively few subtype combinations have been isolated from mammalian species, all subtypes, in most combinations, have been isolated from birds.

• the sudden emergence of antigenically different strains in humans, termed antigenic shift (every 20-40 years) results in a pandemic : historically, flu pandemics have come in two or three waves, lasting a total of 13-23 months. Estimates of R₀ for pandemic influenza strains range from 1.8 to 3 for the 1918 Spanish flu^{ref1, ref2} and are around 1.9 for the H₃N₂ pandemic^ref
• the first documented one occurred in 1580
• 1889-1891 (H₂N_?) : Siberia => Europe
• it has occurred on 4 occasions in the 20^th Century, in .. :
• 1902 (H₃N_?)
• 1918-1919 (H₁N₁) : killed an estimated 40 to 50 million people; 3 peaks of mortality; the second and highest peak occurred 6 months after the initial outbreak
• 1932-1933 (H₁N₁) (A₀)
• 1947-1948 (H₁N₁) (A')
• 1957-1958 (H₂N₂) (Asian) : killed an estimated 2 million people, 70,000 in the USAs alone. The rate of infection with symptomatic flu that year exceeded 50% in urban populations
• 1968 (H₃N₂) (Hong Kong) : killed an estimated 1 million people, 34,000 in the USA
• 1976 (H₁N₁) (swine). In January and February 1976 200 soldiers were infected and 1-2 died at Fort Dix, New Jersey, because of swine flu virus (H₁N₁) (as detected by conversion of serial sera from negative to positive for swine flu hemagglutinins), thought to be a direct descendant of the virus that caused the pandemic of 1918. This conclusion was based on antibodies to H₁N₁ antigens found in survivors of the 1918 pandemic, and the belief that the 1918 virus was eventually transmitted to pigs in the Midwest, where it persisted and caused sporadic human cases. The Public Health Service decided to prepare an influenza vaccine with the Fort Dix strain and immunized 25% of the US population, or 45 million persons, within October, although the predicted pandemic never occurred. On the basis of available historical data, the R₀ of the strain was 1.1–1.2. This number suggests that swine flu would not have become a major epidemic (Neustadt RE, Fineberg HV. The swine flu affair: decision-making on a slippery disease. Washington: US Department of Health, Education and Welfare; 1978; Shilts R. And the band played on: politics, people, and the AIDS epidemic. New York: St. Martin's Press; 1987). Any narrative on the swine flu episode would be incomplete without mentioning the work of Richard Shope on the possible relationship between the putative influenza virus of 1918 and its eventful residence in pigs in Iowa, where it caused an influenzalike syndrome and where it remained as a reservoir (Shope RE. The influenzas of swine and man. In: The Harvey lectures, 1935–1936. New York: The Harvey Society; 1937. p. 183–213). Whatever the merits of this argument about the cause of swine flu virus infection in adults in the 1930s, of interest here is Francis's suggestion that the swine flu antibody in humans was the result of repeated exposure to human strains, and perhaps not due to prior infection with the 1918 virus. Surely his thoughts about this matter were the genesis of the concepts expressed in On the Doctrine of Original Antigenic Sin, published in 1960 (Francis T Jr. On the doctrine of original antigenic sin. Proc Am Philos Soc. 1960;104:572). Francis wrote, "The antibody of childhood is largely a response to dominant antigen of the virus causing the first type A influenza infection of the lifetime. The antibody-forming mechanisms are highly conditioned by the first stimulus, so that later infections with strains of the same type successfully enhance the original antibody to maintain it at the highest level at all times in that age group. The imprint established by the original virus infection governs the antibody response thereafter. This we have called the Doctrine of the Original Antigenic Sin." Francis died in 1969 and did not live to know the full explanations for antigenic shift through reassortment of gene segments from 2 parent viruses or antigenic drift through mutation. He surely would have been in awe, as we all are, of the molecular explanation of influenza virus variation with succeeding epidemics. And yet, even with the brilliant work of Taubenberger delineating the 1918 virus^ref, we can still ask Francis's question: Which strain will cause the next pandemic?
• 1977 (H₁N₁) (Russian); it did not replace the H₃N₂ subtype and both subtypes continued to cocirculate
• next (H₅N₁) (avian) ???
Pandemic influenza may originate through at least 2 mechanisms:
• reassortment between an animal influenza virus and a human influenza virus that yields a new virus :  a key event would probably be a change in the binding specificity of the virus from a receptor in the lower respiratory tract to one in the upper respiratory tract. This may result in a decrease in at least the initial pathogenicity, as the infection would be more likely to start with a tracheal bronchitis rather than pneumonia (see below). In addition, if human adaptation resulted from reassortment with a human virus, pathogenicity factors on gene segments not in the resulting reassortment would be lost, and there may be a degree of prior immunity in the population to the human virus–derived gene segments, both further reducing pathogenicity in humans


• direct spread and adaptation of a virus from animals to humans : pandemic strains of influenza A virus arise by genetic reassortment between avian and human viruses : pigs could serve as "mixing vessels" for such reassortment events since they express both a2-3-linked and a2-6 linked sialic acids on epithelial cells of the lungs and upper respiratory tract (see below)^ref. After the outbreaks of group A subtype H₅N₁ (A/H₅N₁) avian influenza viruses in Hong Kong in 1997, in which 6 people died, the hypothesis was put forward that not only pigs but also humans themselves might serve as mixing vessels for the next pandemic influenza virus^ref. It should be kept in mind also that the "mixing vessel" hypothesis is no more than a hypothesis, and the role of pigs as "mixing vessels" has not been established unequivocally. Genetic reassortment between avian and human influenza A viruses in Italian Sus scrofa occurred at some time between 1983 and 1985 : human-like H₃N₂ strains isolated from 1985 to 1989 contained the internal protein genes of avian-like H₁N₁ viruses, whereas those isolated in 1977 and 1983 did not^ref. Multiple genetic reassortment of avian and human influenza A viruses in European Sus scrofa, resulting in the emergence of an H₁N₂ virus of novel genotype^ref. Predictions for future human influenza pandemics^ref. The evolution of human influenza viruses^ref. Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics^ref. Emergence of influenza A viruses^ref.
The 1918 virus did not originate through a reassortment event involving a human influenza virus: all 8 genes of the H₁N₁ virus are more closely related to avian influenza viruses than to influenza from any other species, indicating that an avian virus must have infected humans and adapted to them in order to spread from person to person^{ref1, ref2}. In both 1957 and 1968, a new influenza virus emerged because of reassortment events involving 2 influenza viruses. The segmented genome allows each influenza A virus to exchange genetic material with other influenza A viruses. In 1957, dual infection of an individual animal — probably a human, but possibly another species, such as a pig — with an avian H₂N₂ influenza and a human H₁N₁ influenza resulted in the emergence of a new influenza virus containing the hemagglutinin, the neuraminidase, and the gene for one of the polymerase proteins (PB1) from the avian virus, along with the remaining 5 genetic segments from the human H₁N₁ influenza virus. The new reassortant virus circulated in humans until 1968, when it was replaced by another reassortant virus, the H₃N₂ Hong Kong virus — created by the replacement of the hemagglutinin (H₂) and polymerase (PB1) genes of the H₂N₂ virus with 2 new avian genes, H₃ and a new PB1. Today, the descendants of this virus continue to cause the majority of influenza infections in humans. 5 of the genes of today's H₃N₂ influenza virus have their origin in the 1918 pandemic.

To investigate how often antigenic shift happens, researchers looked at the published genome sequences of 156 flu strains that circulated in New York state between 1999 and 2004. They constructed family trees that show how these viruses slowly evolved over time. And they discovered at least 4 instances where a virus had picked up a gene from another virus through reassortment^ref. In one of these instances the gene swap resulted in a particularly nasty strain that struck New York in the winter of 2003-04. This 'Fujian' strain was produced when a virus that was common the previous year picked up a gene from a relatively harmless strain, which had been around for some years but infected very few people. The gene was for a protein that the virus uses to bind to cells it is going to infect. The version the dominant strain picked up was very different to the one it had before, catching people's immune systems, and vaccines, unawares. The researchers found a wider variety of strains circulating at any one time than expected, and more instances of gene swapping. Multiple lineages coexist at the same time and place, and the key thing is that they are reassorting and doing so quite frequently. Flu researchers already had a good idea of how reassortment happens, from studies that tracked specific genes for surface proteins, but Holmes's research goes beyond this by using whole genome sequences, giving a clearer picture of how often this happens. These analyses will help us understand in detail the evolution of the influenza virus, taking into account more than just these genes. Further studies investigating the genome sequences of bird-flu viruses could help us understand whether recombination is as just common in those. Some researchers are now trying to create recombinant strains in the lab, using bird and human viruses, to see what a pandemic virus might look like and how easily it might form. The longest inter-pandemic interval recorded with certainty is 39 years. It is not possible to know whether the current H₅N₁ is capable of adapting to humans so that it can spread with high efficiency through low-titer aerosol transmission to initiate an influenza pandemic. However, Taubenberger et al. provide some guidance on the genetic changes that might be required for such an event. The role of PB1 must be critical, since in both 1957 and 1968, this polymerase gene was transferred along with the hemagglutinin during reassortment. By comparing the consensus sequence of the 3 avian influenza polymerase genes PA, PB1, and PB2 with the 1918 sequence, as well as with more contemporary influenza viruses, Taubenberger et al. have identified 4 amino acids of PA, one of PB1, and 5 of PB2 that are found in human influenza viruses (including the 1918 virus) but generally not in avian influenza viruses. In 2 instances, these amino acids are found in nuclear localization signaling (NLS) regions, suggesting that some or all of these amino acid differences are critical for the virus to adapt to humans. The genetic sequences of the 1997 Hong Kong H₅N₁ virus and the 2004 Vietnam H₅N₁ virus reveal that several human isolates of these viruses contain 1 of the 5 amino acid changes in PB2 that have been identified as important to the ability of the 1918 virus to infect humans. This finding suggests that several additional genetic changes must occur before these viruses will begin to spread efficiently from person to person. The genetic sequences of avian viruses may provide a window through which to monitor these sporadic transmissions for the potential of the viruses to adapt to humans. The occurrence of additional genetic changes in the avian H₅N₁ virus circulating in birds that match the consensus sequence for PA, PB1, or PB2 in human influenza would be cause for heightened concern. On the basis of the rates of replacement of amino acids, Taubenberger et al. estimated that avian influenza polymerase genes had been circulating in humans as early as 1900. If this estimate is correct, then monitoring of the sequences of viruses isolated in instances of bird-to-human transmission for genetic changes in key regions may enable us to track viruses years before they develop the capacity to replicate with high efficiency in humans. Knowledge of the genetic sequences of influenza viruses that predate the 1918 pandemic would be extremely helpful in determining the events that may lead to the adaptation of avian viruses to humans before the occurrence of pandemic influenza. We could then conduct worldwide surveillance for similar events involving contemporary avian viruses^ref. Deaths occurred predominantly in infants and the elderly during the 1889 and 1947 pandemics, while young adults were most severely affected in 1918. Pandemic stages, as defined by the WHO in May 2005 :
• interpandemic period :
• phase 1 : no new influenza virus subtypes have been detected in humans. An influenza virus subtype that has caused human infection may be present in animals. If present in animals, the subnational levels. risk a of human infection or disease is considered to be low.
• phase 2 : no new influenza virus subtypes have been detected. However, a circulating animal influenza virus subtype poses a substantial risk a of human disease.
• pandemic alert period :
• phase 3 : human infection(s) with a new subtype, but no human-to-human spread, or at most rare instances of spread subtype and early detection, notification to a close contact.
• phase 4 : small cluster(s) with limited human-to-human transmission but spread is highly localized, suggesting that the virus is not well adapted to humans.
• phase 5 : larger cluster(s) but human-to-human spread still localized, suggesting that the virus is becoming increasingly better adapted to humans, but may not yet be fully transmissible (substantial pandemic risk).
• pandemic period :
• phase 6 : pandemic: increased and sustained transmission in general population
• frequent epidemics have occurred between the pandemics as a result of gradual antigenic change in the distal region of HA of the prevalent virus, termed antigenic drift (every 2-3 years). Antigenic differences among viral isolates are measured by the hemagglutination inhibition (HI) assay, but these data are sometimes difficult to interpret. Antigenic cartography resolves many of these difficulties, increases the resolution at which antigenic data can be interpreted, makes the interpretation more quantitative, and provides a visualization of antigenic differences among strains^ref. These methods are now integrated into the biannual WHO seasonal vaccine strain-selection process. They are also being used to map the antigenic evolution of H₅ viruses and will be used to evaluate the breadth of immunity offered by pandemic vaccines. Currently, epidemics occur throughout the world in the human population due to infection with influenza A viruses of subtypes H₁N₁ and H₃N₂ or with influenza B virus : each year an ordinary flu epidemic causes approximately 250,000-1,000,000 deaths worldwide (36,000 deaths and 114,000 hospitalizations in USA and 4,000 deaths in UK) : > 90% of deaths occur among people aged > 65. The circulating viral subtype is associated with varying severity of influenza epidemics^ref : in the last 2 decades in the USA, estimated excess death rates were on average 2.8-fold higher in A/H₃N₂-dominated seasons than in A/H₁N₁ and B seasons^ref. Within a given subtype, however, the strain-specific determinants of epidemic severity are still poorly understood. For instance in the USA in the same period, excess death rates varied nearly 4-fold among A/H₃N₂ seasons, even after adjustments for population aging^ref. During 1990 to 1999, about 92 influenza-related deaths occurred annually among children aged <5 years in the USA^ref. > 18,000 (>90%) of these deaths and approximately 48,000 of the pneumonia and influenza hospitalizations per year occur among persons who are at highest risk for influenza-related complications. Children aged < 5 and women in the third trimester of pregnancy are also at higher risk for serious complications. Annual influenza epidemics have resulted in an average of >18,000 excess deaths (i.e., the number of influenza-related deaths above a projected baseline of expected deaths) and 48,000 pneumonia and influenza hospitalizations among older persons in the USA. Influenza season typically peaks in the USA between December and March. An analysis of data collected by the European Influenza Surveillance Scheme (EISS) during the past 5 winters (1999 to 2004) reveals a possible west-east spread of influenza across Europe^ref. In 3 of the 5 winters (2003/2004, 2002/2003, 2001/2002), the analysis suggests that there was west-east spread and during one of these seasons (2001/2002) there was also a south-north spread. clinical activity (usually cases of influenza-like illness (ILI), but occasionally cases of acute respiratory infection) reported by sentinel physicians and collected by EISS is a valid indicator of influenza activity and that, for Europe as a whole, increased influenza activity lasts for 10 to 22 weeks (2 to 5 months) each season^ref. West-east spread is counter to the prevailing notion that flu outbreaks spread East-west to Europe from Asia. Anecdotal accounts exist in the literature of historical influenza epidemics associated with unusual numbers of deaths, such as occurred in the 1951 epidemic in England in the midst of the first era of A/H₁N₁ viruses (1918–1957) (Burnet M. The influenza virus. Scientific American. 1953;188:28–31). In Liverpool, where the epidemic was said to originate, it was "the cause of the highest weekly death toll, apart from aerial bombardment, in the city's vital statistics records, since the great cholera epidemic of 1849"^ref. This weekly death toll even surpassed that of the 1918 influenza pandemic : the 1951 influenza epidemic had greater death rate than all subsequent influenza epidemics or pandemics in England and Canada. But what sets the 1951 epidemic apart from pandemics is that the older population was also severely affected, with twice the deaths as occurred in the 1957 pandemic. By contrast, the 1951 epidemic had minor impact in the USA, except possibly in New England. Laboratory surveillance data from the World Health Organization (WHO) indicate that influenza A viruses circulating at the time were characterized as H₁N₁^ref, a subtype circulating since the 1918 pandemic^ref. Although an unusually large drift event in the hemagglutinin of A/H₁N₁ viruses was reported in 1947^ref, subsequent changes in this protein remained minor until after 1951^ref. Hence, no virologic evidence of a shift or unusual drift in the hemagglutinin antigen exists for 1951 viruses. In support of the virologic evidence, we have shown that no epidemiologic pandemic signature occurred in 1951, as indicated by an age shift of deaths towards younger age groups^ref. The 1951 epidemic exhibited geographic disparities in influenza-related deaths, as illustrated by the contrast between England and Canada (countries with high death rate) and the United States (low death rate). These disparities are in part explained by laboratory surveillance reports by WHO^{ref1, ref2}, indicating that 2 antigenically distinct influenza A/H₁N₁ strains cocirculated in the Northern Hemisphere during the 1951 epidemic^{ref1, ref2}. The so-called Scandinavian strain was isolated in northern Europe and associated with mild illnesses. By contrast, the "Liverpool strain" was associated with severe illnesses and high deaths in Great Britain, Canada, southern Europe, and Mediterranean countries^ref. As both strains cocirculated in some countries^ref, intrasubtypic cross-immunity might have existed, with these 2 strains competing for susceptible hosts. The precise reasons for the unusually high death rate associated with the Liverpool strain remain elusive. The genetic markers of influenza virulence are still unclear today, but a multibasic cleavage site in the hemagglutinin, as well as minor changes in internal genes, are believed to enhance viral pathogenicity^ref (Nicholson K, Hay A. Textbook of influenza. Oxford: Blackwell; 1998). Only hemagglutinin inhibition tests could be performed in 1951, and to our knowledge, no influenza virus isolate or genetic sequence from 1951 is available in the public domain. Further molecular analysis of 1951 influenza specimens could help explain the extreme local pathogenicity in that season. We have described an influenza season that was unexpectedly severe in some countries and mild in others. This geographic disparity in influenza-related deaths is not common; influenza mortality is generally correlated between the USA and Europe and within the USA^{ref1, ref2}. Occasional disparities have been reported, however. For instance, the impact of the 2 waves of the 1968 pandemic differed markedly between North American and Eurasian countries, perhaps because of differences in preexisting immunity and evolving viruses^ref. In this context, the 1951 epidemic appears as another striking example of geographic disparities in influenza impact, perhaps explained in this case by cocirculation of 2 influenza A/H₁N₁ strains. Other competing hypotheses include differences in preexisting population immunity or socioeconomic factors, but these are less parsimonious explanations^ref

• interhuman : 7 (H) subtypes (H₁, H₂, H₃, H₅, H₇, H_{9, and H}) and 3 (N) subtypes (N₁, N₂ and N₇) have been isolated in the past from humans
• both influenza A and B viruses survive for 24-48 hr on hard, nonporous surfaces such as stainless steel and plastic but survive for < 8-12 hr on cloth, paper, and tissues. Measurable quantities of influenza A virus are transferred from stainless steel surfaces to hands for 24 hr and from tissues to hands for up to 15'. Virus survives on hands for up to 5' after transfer from the environmental surfaces. These observations suggest that the transmission of virus from donors who are shedding large amounts could occur for 2-8 hr via stainless steel surfaces and for a few minutes via paper tissues. Thus, under conditions of heavy environmental contamination, the transmission of influenza virus via fomites may be possible^ref.
• cross-species :
• some strains can be passed from humans to Mustela putorius furo.
• birds : 16 haemagglutinin (H) and 9 neuraminidase (N) subtypes of Influenza A viruses have been isolated from birds;
• avian influenza (AI) has been detected in Sus scrofa :
• WU Rui, et al. Isolation and identification of swine influenza virus. Virologica Sinica 2003; 18(6): 553-6.
• Li Hai-yan, et al. Isolation and characterisation of H₅N₁ and H₉N₂ influenza viruses from pigs in China. Chinese Journal of Preventive Veterinary Medicine 2004; 26(1). During 2002-2003, 1936 sera samples were collected from the pig flocks of 14 provinces and cities for H₉ subtype influenza detection. 7.3, 6.8, 4.5 and 1.9% seropositivity rates against avian H₉ subtype virus were detected in serum samples collected in 2002 from Liaoning, Guangdong, Shandong provinces and Chongqing city, respectively. H₉ subtype was not detected from serum samples collected in 2003; but 4.7 and 8.2% of serum samples collected from Guangdong and Fujian provinces were H₅ subtype influenza positive. 6 H₉N₂ and 2 H₅N₁ subtype influenza viruses were isolated from the samples that were collected during 2001-2003. Sequence analysis confirmed that these viruses are closely related to the H₉N₂ or H₅N₁ subtype avian influenza viruses that have been isolated in China. Serological tests (apparently carried out in 2003?) showed that the % of the positive sample for H₅ was 35.0 (7/20), 23.8 (5/21), and 20.0 (3/15) for 3 swine farms tested, respectively. 2 isolates of the A H₅N₁ virus strain were obtained from pigs, the 1st one in 2001, named SIV SW/FJ/F1/2001. There is no mention of the kind of tested sample, which could be nasal swab, or tissue, or both. The 2nd isolate was obtained in 2003, named SIV SW/FJ/1/03, from a nasal swab. Obviously, both originated in the province of Fujian, though the exact location is not mentioned. However, 3 locations are mentioned in the acknowledgements: Nanping city, Fuqing city, and Putian city, all located in Fujian Province. It would be interesting to note the origin (nasal swab or infected tissue?) from which the recent isolate(s) was/were obtained. One H₅N₁ strain was isolated in 2002 from materials collected from pigs in the Fujian province in 2001. Subsequently, 1936 samples were collected in 2003 from pigs in 14 provinces including Fujian and again one H₅N₁ strain was isolated from that province. These 2 isolates have been subjected to detailed analysis and have been shown to be highly homologous to the duck-derived H₅N₁ virus isolated recently in birds in China. According to the Chinese authorities, no variation in the virus has been observed. More recently, as part of their ongoing epidemiological surveillance programme, 1.1 million samples including 4447 from pigs have been collected and analysed between April and August 2004 from 10 provinces including Fujian, and no infection from H₅N₁ has been detected in pigs. The above findings do not at this stage indicate any major evolution regarding H₅N₁ infection in pigs. However, the OIE wishes to insist on the necessity for Member Countries affected by avian influenza to increase their epidemiological surveillance to better understand the implications of the existence of H₅N₁ virus in pigs. This study confirms the H₉N₂ influenza infection in pig flocks in China, and also is the 1st report of the emergence of the H₅N₁ influenza virus in the pig species. Therefore, for both the veterinary and public health sectors, urgent attention should be paid to the pandemic preparation for these 2 influenza subtypes. The importance of the findings is difficult to assess since the samples were obtained during the period 2001-2003 and the information has only now been disclosed. The frequency of the H₅N₁ virus seems to be very low and occasional infection of pigs (not to mention humans) may occur infrequently without onward transmission of virus
• A/Swine/Shandong/1/2003(H₉N₂) probably originates from a reassortment of chicken influenza virus subtype H₉N₂, H₅N₁ and duck influenza virus subtype H₉N₂. Although A/Swine/Shandong/1/2003(H₉N₂) differs from human isolates A/Hongkong/1073/99(H₉N₂) and A/Hong Kong/1074/99(H₉N₂), we should still pay more attention to the prevalence of the H₉N₂ subtype^ref.
• neutralizing antibodies to H₁, H₃, H₄, and H₅ influenza viruses were detected in the serum samples collected in 1977­1982 and 1998, suggesting that pigs in China have been sporadically infected with avian H₄ and H₅ viruses in addition to swine and human H₁ and H₃ viruses^ref.
• preliminary virological and serological evidence of H₅N₁ virus infection of pigs in Fujian province have been obtained^ref.
• XU Chuan-tian, et al. Isolation and Identification of type A swine influenza virus from Shandong Province and sequence analysis of HA gene. Virologica Sinica 2004; 19(1): 27-31.

The Fujian strain of influenza A (H₃N₂) virus appears to be the currently predominant strain in the USA and Europe. The Panama strain, which is a constituent of the current vaccine, is responsible for the current outbreak in western Canada. Although there is believed to be sufficient antigenic cross-reactivity between the Fujian and Panama strains for the existing vaccine to be protective, it has been judged prudent to replace the Panama strain with the Fujian strain in the new Southern Hemisphere vaccine, although the Panama strain is still in circulation in some places.
The mouse has been a valuable and extensively used model to study the mechanisms that protect against or promote recovery from this infection. Evidence indicates that components of both innate^{ref1, ref2, ref3} and adaptive^{ref1, ref2} immune systems contribute to the control of the infection and that activities provided by CD4⁺ helper (T_h) and B cells^{ref1, ref2} or CD8⁺ cytotoxic T (T_c) cells^{ref1, ref2, ref3} can independently resolve it, although the latter are generally believed to be more effective. The recovery process mediated by T_h and B cells appears to depend largely on the generation of a T_h-dependent antiviral Ab response, as neither T_h^{ref1, ref2, ref3} nor B cells^ref are capable of resolving the infection on their own, while infection in SCID mice can be cured by treatment with Abs specific for the viral hemagglutinin (HA) molecule^{ref1, ref2}. The high therapeutic efficacy of these Abs appears to be due to their ability to concomitantly suppress yield of progeny virus from infected cells and prevent released progeny virus to spread the infection to new host cells^{ref1, ref2}. The T_c cell-mediated recovery process has been shown to rely mainly on the perforin/granzyme- and Fas-mediated killing of infected host cells^{ref1, ref2}, while secretion of cytokines such as IFN-g, which may inhibit virus spread by inducing cellular resistance to infection, does not appear to play a significant role^{ref1, ref2}, at least in the intact mouse^ref, but may become important if the Tc activity is being tested at its therapeutic threshold^ref. The above implies that effector T_c (eT_c) are capable of killing infected epithelial cells before the release of progeny virus. This is surprising in the case of an acute infection in which the eTc has available only a short window of time (between expression of viral peptides by MHC class I and release of virions) to perform this task^{ref1, ref2}. The massive recruitment of virus-specific eT_c into the cellular exudate of the infected airways at the time of virus clearance would be consistent with such a scenario^ref. However, since evidence for the autonomy of T_c-mediated clearance was obtained in the study of influenza viruses of low pathogenicity, such as X31^{ref1, ref2} and B/AA^ref, we wondered whether eT_c-mediated control mechanisms would be similarly effective also against a more pathogenic, and perhaps more rapidly replicating, influenza virus strain like PR8. There is evidence from other virus systems that rapidly replicating viruses such as vesicular stomatitis virus and Semliki Forest virus or more virulent variants of lymphocytic choriomeningitis virus are not effectively controlled by T_c^{ref1, ref2, ref3, ref4, ref5}. In addition, 2 of the influenza virus studies^{ref1, ref2} had been done in mice that contained B cells, although no T_h cells. Therefore, the conclusion that T_c resolved the infection autonomously in these mice assumed that the B cells made no significant contribution to recovery without help from T_h cells. B cell-deficient mice (µMT) of BALB/c background additionally depleted of T_h cells by chronic treatment with anti-CD4 Ab GK1.5 were used to test the ability of the T_c response to autonomously resolve the highly pathogenic PR8 and the less pathogenic X31 virus infections : the study confirmed that the T_c response has the basic capability to autonomously (in conjunction with innate defense) resolve these infections, but with substantial delay compared with immunologically intact mice, which resulted in high mortality in infection with the pathogenic PR8 strain. The study further showed that B cells contributed to the recovery process by a T_h-independent mechanism of still undefined nature^ref.
However, in contrast to findings in mice, the protective value of memory Tc cells in humans remains controversial. The classic study by McMichael et al.^ref indicated that presence of memory Tc cells in blood, which could give rise to Tc cells on stimulation in vitro, correlated with reduced virus shedding 3–4 days after volunteers were challenged with a wild-type virus, but had no significant effect on illness. Subsequent studies performed in children found no significant difference in shedding of attenuated vaccine strains in patients who had recovered from previous infection with a vaccine or natural strain of a different subtype than did study participants who had no evidence of previous virus exposure^ref (Wright PF, Johnson PR, Karzon DT. Clinical experience with live, attenuated vaccine in children. In: Options for the control of influenza;1986. New York: Alan R Liss, Inc. p. 243–53). Similarly, children vaccinated with an H₁N₁ strain showed no difference in attack rate and febrile respiratory illness during exposure to natural epidemic H₃N₂ virus from controls who received a placebo^ref

The pathogenesis of influenza infections has been associated with alteration in the lymphohemopoietic system^{ref1, ref2, ref3, ref4, ref5,ref6, ref7} (20, 21, 29, 31, 50-52). Experimental infection of chickens with the avian influenza virus A/Turkey/Ontario/7732/66 (H₅N₉) (Ty/Ont) resulted in the destruction of lymphocytes and histopathological necrosis of lymphoid tissues. It was further demonstrated that the lymphocyte destruction in birds was associated with virus-induced apoptosis, as Ty/Ont, but not a human strain, A/Puerto Rico/8/34 (H₁N₁), induced apoptosis of an avian lymphocyte cell line^ref. Whether an avian virus has an affinity for cells of the mammalian immune system, resulting in leukocyte death, remains an unanswered question. Infection of mice with a highly virulent H₅N₁ resulted in a decrease in peripheral blood and tissue lymphocytes and aberrant cytokine and chemokine production. An increase in apoptotic cells in the spleen and lung tissue is identified as a possible cause of lymphocyte death.

A variety of influenza A viruses (avian, equine, swine, and human), as well as human influenza B viruses, induced DNA fragmentation in a permissive mammalian cell line, Madin-Darby canine kidney (MDCK), and this correlated with the development of a cytopathic effect during viral infection. Since the proto-oncogene bcl-2 is a known inhibitor of apoptosis, we transfected MDCK cells with the human bcl-2 gene; these stably transfected cells (MDCKbcl-2) did not undergo DNA fragmentation after virus infection. In addition, cytotoxicity assays at 48 to 72 h after virus infection showed a high level of cell viability for MDCKbcl-2 compared with a markedly lower level of viability for MDCK cells. These studies indicate that influenza A and B viruses induce apoptosis in cell cultures; thus, apoptosis may represent a general mechanism of cell death in hosts infected with influenza viruses^ref

Influenza virus has been shown to induce apoptosis in tissue culture cells^{ref1, ref2} and in peripheral blood monocytes^{ref1, ref2}. A depletion of lymphocytes due to apoptosis has also been described in mice infected with a highly virulent influenza A virus (IAV) (H₅N₁) isolated from humans^ref. The immunopathological mechanisms and the role played by the virus infection of leukocytes with respect to disease pathology in general and leukocyte death in particular have not been elucidated. An early lymphopenia has been described in IAV-infected patients^{ref1, ref2, ref3}, and inoculation of humans with IAV has been shown to cause a decrease in both T- and B-cell numbers during illness^{ref1, ref2}. In the experimental infections, volunteers developed a severe T-cell lymphopenia and a moderate B-cell lymphopenia even though seroconversion occurred in 90% of the volunteers, suggesting that T- and B-cell functions were preserved^{ref1, ref2}. This observed lymphopenia could be the result of cell migration from the circulation and/or cell death caused by necrosis or by apoptosis or through suppression of hematopoeisis. Lymphocyte depletion via apoptosis after exposure to IAV could be the result of virus-induced cytokine stimulation, viral induction of Fas, or other cell-virus interactions^ref.

Among leukocytes, only monocytes and macrophages were found to be highly susceptible to an infection by influenza A virus. After infection, de novo viral protein synthesis was initiated but then interrupted after 4-6 h. Most macrophages died by apoptosis within 25-30 h. Before cell death, however, macrophages responded to influenza A virus with a high cytokine gene transcription and subsequent release of TNF-a, IL-1, IL-6, IFN-a/b, and CC-chemokines. The basic mechanisms of virus-induced cytokine expression are still unknown and appear to involve transcription factors such as NF-kB and AP-1 which, however, were only activated for 2 h and declined below control values thereafter. After influenza A virus infection, only the mononuclear cell attracting CC-chemokines MIP-1a, MIP-1b, and RANTES were produced while the prototype neutrophil CXC-chemoattractants IL-8 and GRO-a were entirely suppressed. This selective induction of CC-chemokines may explain the preferential influx of mononuclear leukocytes into virus-infected tissue. Monocytes and macrophages represent a primary target for an influenza A virus infection. Thus, the mononuclear phagocyte response leads to a rapid proinflammatory reaction and an enhanced immigration of mononuclear leukocytes, which may condition the infected host for the subsequent virus antigen-specific defense^ref

During acute illness^ref or induced infection^ref, lymphopenia is evident as reduced numbers of B and T cells. This may reflect migration of lymphocytes to the site of infection, and it would therefore be reasonable to expect that lymphopenia would correlate with recovery from infection. However, in the recent influenza A virus H₅N₁ outbreak, low leukocyte counts correlated with severity of disease^ref. In addition, the T cells present during acute infection are functionally impaired, with reduced lectin-induced stimulation^{ref1, ref2}, suggesting that these quantitative and qualitative changes may not simply be due to migration of cells. A number of factors probably contribute to these observations. For example, virus load, as well as viral components that confer pathogenicity, may influence the milieu of cytokines and the composition of responding cells. These factors may act on both naïve and effector B and T cells to result in lymphopenia. The cell type that may mediate this lack of response is the dendritic cell (DC), since it transports virus to the draining lymph node^ref and has direct contact with T cells. The interactions between DC and naïve and memory T cells determine both the magnitude and quality of the immune response. Influenza virus alters this interaction in vitro^ref. In this in vitro system, we examined the effects of influenza virus infection on DC function. DC were cultured from H-2^b bone marrow and then used to stimulate H-2^d allogeneic T cells. Since this response is not virus specific, the ramifications of influenza virus infection were determined by comparing T-cell proliferation stimulated by uninfected and virus-infected DC. When DC were infected with a low dose of A/PR/8/34 (PR8), there was increased T-cell proliferation in response to influenza virus-infected DC^ref. This altered response was dependent on viral neuraminidase (NA) and did not require infection of the DC with influenza virus. One or more mechanisms may mediate this effect when sialic acid is removed from glycoconjugates at the DC surface. This may include changes that facilitate interactions between the major histocompatibility complex (MHC) class I-peptide complex with the TcR, B7-1 with CD28, and adhesins with their ligands or changes in charge at the cell's surface that result in a general increase in contact. However, our current results show that this enhanced proliferative response is not observed when DC are infected with high doses of PR8. There may be multiple reasons for the lack of an enhanced response at high PR8 multiplicity of infection (MOI). For example, since influenza virus induces apoptosis of infected cells^ref, greater numbers of virus particles may induce greater DC apoptosis, thereby reducing the number of effective stimulators in the culture. Alternatively, at high doses of virus, virions released from the DC may interact with T cells, resulting in their reduced proliferation. Viral NA could contribute to this reduced response by desialylation of T-cell surface glycoproteins. This would result in DC and T cells having equal charges, so that opposite attractive charges would no longer facilitate the interaction between them. Other reasons for the dose-dependent proliferative response may be that properties of DC that contribute to successful T-cell activation are altered at high virus doses, or that, under these conditions, cytokines that inhibit proliferation are secreted. At a high MOI, a number of changes occur in DC. The most notable physical change that provides a reasonable mechanism to explain the reduced response to DC infected with high doses of PR8 is the formation of DC clusters that exclude T cells. However, neutralization of TGF-b₁ restored the enhanced alloreactive T-cell response, suggesting that this cytokine plays a primary role in reducing proliferation^ref.

• human SAP binds much more avidly than mouse SAP to the virus, but has no effect  in human influenza infection.
• clonal characteristics of effector CD8⁺ T cells specific for :
• in HLA-A2⁺ adults is almost exclusively directed at residues 58–66 of the virus matrix protein (MP(58–66)). Vb17Va10.2 TCRs containing a conserved arginine-serine-serine sequence in CDR3 of the V segment dominate this response. Whereas the TcR typically fits over an exposed side chain of the peptide, in this structure MP(58–66) exposes only main chain atoms. This distinctive orientation of Vb17Va10.2, which is almost orthogonal to the peptide-binding groove of HLA-A2, facilitates insertion of the conserved Arg in Vb CDR3 into a notch in the surface of MP(58–66)–HLA-A2.
• nucleoprotein (NP) and acid polymerase (PA) peptides presented by H2D^b. Using a single-cell approach and determination of CDR3b profiles, a limited, predominantly "public" repertoire was found for CD8⁺D^bNP₃₆₆⁺Vb8.3⁺ cells, whereas diverse and "private" TcRb clonotypes were typical of the CD8⁺D^bPA₂₂₄⁺Vb7⁺ response. This single-cell approach has now been used to relate the contributions of particular clonotypes (or affinities) to high-avidity TCRs, as defined by binding under conditions of limiting tetramer availability. At least by the measure of CDR3b usage, no difference could be found between total and high-avidity populations in the spectrum of TCR-pMHC affinities throughout the limited, and relatively public, CD8⁺D^bNP₃₆₆⁺Vb8.3⁺ populations. Conversely, the more even (by clone size), diverse, and private CD8⁺D^bPA₂₂₄⁺Vb7⁺ response was characterized by the clear partitioning of the largest T cell clones in the high-avidity compartment. These results suggest that the relatively constrained CD8⁺D^bNP₃₆₆⁺Vb8.3⁺ set utilizes a relatively narrow range of affinities, whereas the broader CD8⁺D^bPA₂₂₄⁺Vb7⁺ response is induced at a range of TCR-pMHC affinities. Thus, whereas TCR sequence (or affinity) appears to contribute substantially to the avidity profile of diverse virus-specific CD8⁺ populations, other mechanisms may be prominent where the TCR spectrum is more limited^ref
• high levels of serum IFN-a are correlated with febrile seizures in pediatric influenza
• MIF and and CXCL8 are released from lung epithelial cells rendered necrotic by influenza A virus infection.

myxovirus resistance-1 (MX1) is a member of the dynamin family of large GTPases induced by type I interferon in neutrophils that self-assembles into long filamentous homo-oligomers then rearranged into rings and more compact helical arrays that tubulate lipids in vitro, associates with the smooth endoplasmic reticulum, and promotes apoptosis of infected cells (on the contrary of MX2)

• observing live influenza virus in human serum or plasma is unusual. In 1963, low quantities of virus were isolated from blood of a patient on day 4 of illness^ref, and in 1970, the virus was cultivated from blood specimens from 2 patients^ref. Recently, a fatal case of avian influenza A (H₅N₁) in a Vietnamese child was reported. The diagnosis was determined by isolating the virus from cerebrospinal fluid, fecal, throat, and serum specimens^ref; viral RNA was found in 6 of 7 serum specimens 4–9 days after the onset of illness^ref. In this case, the H₅N₁ virus could be isolated from plasma on day 10 after symptoms developed. A titer of 3.08 × 10³ copies/mL was obtained from the plasma of a 5-year-old Thai boy with H₅N₁^ref. This case showed the virus in the patient's blood, which raises concern about transmission among humans. Because probable H₅N₁ avian influenza transmission among humans has been reported^ref, this case should be a reminder of the necessity to carefully handle and transport serum or plasma samples suspected to be infected with H₅N₁ avian influenza. Because viable virus has been detected in blood samples, handling, transportation, and testing of blood samples should be performed in a biosafety (category III) containment laboratory to prevent the spread of the virus to healthcare and laboratory workers.

anorexia

photophobia

• M2 inhibitors : the largest proportion of Asian amantadine-resistant H₅ and H₉ AIV during 1991-2004 occurred in China : resistance is spreading more rapidly among avian influenza viruses of H₅N₁ subtype in Southeast Asia than in North America (Virology). Adamantanes have been used to treat influenza A virus infections for many years. Studies have shown a low incidence of resistance to these drugs among circulating influenza viruses; however, their use is rising worldwide and drug resistance has been reported among influenza A (H₅N₁) viruses isolated from poultry and human beings in Asia. We sought to assess adamantane resistance among influenza A viruses isolated during the past decade from countries participating in WHO's global influenza surveillance network. We analysed data for influenza field isolates that were obtained worldwide and submitted to the WHO Collaborating Center for Influenza at the US Centers for Disease Control and Prevention between 1 Oct 1994, and 31 Mar 2005. Pyrosequencing, confirmatory sequence analysis, and phenotypic testing were used to detect drug resistance among circulating influenza A H₃N₂ (n=6524), H₁N₁ (n=589), and H₁N₂ (n=83) viruses. > 7000 influenza A field isolates were screened for specific amino acid substitutions in the M2 gene known to confer drug resistance. During the decade of surveillance, a significant increase in drug resistance was noted, from 0.4 % in 1994-1995 to 12.3% in 2003-2004. This increase in the proportion of resistant viruses was weighted heavily by those obtained from Asia, with 61% of resistant viruses isolated since 2003 being from people in Asia. These data raise concerns about the appropriate use of adamantanes and draw attention to the importance of tracking the emergence and spread of drug-resistant influenza A viruses^{ref1, ref2}. Adamantane resistance in US H₃N₂ isolates^{ref1, ref2, ref3, ref4} : 7 of 8 resistant variants (accounting for 98.2% of the total) contained Ser31Asn mutation in M2 protein
• 1992-1995 : 0.8%
• 1996-1997 : 0.4%
• 1998-1999 : 2.2%
• 2000-2001 : 1.4%
• 2002 : 1.4%
• 2003 : 1.7%
• 2004 : 1.9%
• October 2004-March 2005^ref : 14.5%
• October-December 2005 : 92.3%. 3 influenza A(H₁N₁) viruses have been tested and have demonstrated susceptibility to adamantanes; CDC recommended not to prescribe them and to use NAIs^ref
From 1998 to 2004, resistance was observed in 2 of 589 H₁N₁ viruses collected worldwide (0.3%, Val27Ala and Glu34Lys) and in 1 of 82 H₁N₂ viruses isolated in the USA (Ala30Thr). 6 H₃N₂ viruses with Ser31Asn also contained Val27Iso in M2; 2 of 8 H₁N₁ isolates had Ser31Asn.
• neuraminidase inhibitors (NAIs) (when administered within 48 hours of symptom onset, antiviral treatment of influenza can reduce the duration of illness by approximately 1 day in healthy adults. Data on the use of any of the 4 antiviral agents during pregnancy are not available).

• killed and subunit vaccines. The composition of the vaccine is changed each year in response to antigenic shifts and changes in prevalence of influenza virus strains; the vaccine is usually bivalent or trivalent, containing 1-2 influenza virus A strains and 1 influenza virus B strain. Annual immunization before November is recommended for high-risk individuals (persons over 65 years of age and persons with chronic disease). The last time the vaccine missed the mark was in 1997, when the strain A-Sydney appeared that was significantly different from the strain included in the flu shot, which remained just 30-50% protective.
• chemoprophylaxis : inhibitors of coreceptor binding and fusion(amantadine, rimantadine), neuraminidase inhibitors (oseltamivir) can be used for control of institutional influenza outbreaks and are approximately 70-90% effective in preventing illness in healthy adults
• post-exposure prophylaxis : PEP with oseltamivir is likely to be a cost-effective strategy for family contacts in the UK from a healthcare payer perspective when influenza-like illness contact attack rates are 8% or higher and the only treatment given is 'usual care'.^ref

• respiratory tropism
acute laryngitis and epiglottitis
acute bronchitis
bronchiolitis
primary viral interstitial pneumonia
secondary bacterial community acquired bronchopneumonia (e.g. CA-MRSA) after 4-5 days (leading cause of flu death) : ARDS, hemoptysis
• myocarditis :
• influenza A virus-induced rhabodmyolysis and acute congestive heart failure after high-dose therapy and hematopoietic stem cell transplantation for malignant lymphoma. Early treatment with neuraminidase inhibitor (oseltamivir 150 mg/day for 5 days) may improve the clinical outcome^ref
• neurotropism (diagnosis : PCR on CSF)
• postinfectious encephalomyelitis (PIE) (influenza-associated encephalopathy with with bilateral thalamic necrosis^{ref1, ref2} has mostly been reported in Japan) among young children and school-aged children, including adolescents.
• Reye's syndrome (aspirin and other salicylate-containing medications should be replaced by paracetamole for children with fever and respiratory illness)
• radiculitis
• neuritis
• pharyngotonsillitis
• acute liver failure
• sudden deaths associated with influenza in children occurs with atypical symptoms (e.g., abdominal pain, absence of fever, and mild respiratory symptoms)