Influenza is a myxovirus belonging to the family of viruses known as Orthomyxoviridae. The virus was originally confined to aquatic birds, but it made the transition to humans 6000–9000 years ago, coinciding with the rise of farming, animal husbandry and urbanisation.流感是一種黏液病毒屬於正黏液病毒屬（family),本只局限在水禽、但在六至九千年前傳播至人類，正好和農耕、畜牧及都市化的時間點吻合。 These changes in human behavior and population density provided the ecological niche that enabled influenza, as well as a number of other infectious agents such as the viruses that cause measles and smallpox, to move from animals and adapt to a human host.人類行為的改變、人口的集中提供病毒、像流感、麻疹、天花、從動物變成可適應、感染人類。 Influenza as a disease has been recognised for centuries, even though the viruses which cause it were not correctly identified until the early 1930s, first in the UK and then in the USA. 流感數世紀以來廣為認知，但真正證實是病毒致病則是在1930年代的英國。Indeed the name itself is derived from an Italian word meaning ‘influence’, and reflected the widespread belief in medieval times that the disease was caused by an evil climatic influence due to an unfortunate alignment of the stars.流感英文為influenza,是源自義大利文，意思是影響、在中古世紀認為是因星球不正常的排列、造成惡劣氣候的影響。 Our current understanding – that infectious diseases are caused by infectious agents – is so ingrained that such mystical causes for an illness now seem absurd.現在看來這種想法當然荒謬。 However, even during the Middle Ages, people had a sound idea of infection and realised that some diseases could be passed from one individual to another and others could not. For example, the use of quarantine for a disease such as plague, but not for many other illnesses, shows that people could distinguish infectious diseases from non-infectious diseases even if the causative agent and the method of transmission were obscure.但即使在中古世紀、人們也認知到有些疾病會一個傳一個 The idea that influenza is caused by the influence of the stars, though not a satisfactory explanation of how the disease spread, does identify an important feature of flu – that serious epidemics of the disease occur at irregular intervals. For example, in the twentieth century there were at least five major epidemics of flu that spread around the world (a pandemic), and there were less serious epidemics in most years. In times when people believed in the spontaneous generation of life, the stars would have seemed a reasonable explanation for unpleasant and unexpected epidemics. 1.1 Defining influenza How would you define ‘influenza’? Reveal answer You may well have defined influenza as an infection caused by an influenza virus. However, you may have defined it according to its symptoms: an infection that starts in the upper respiratory tract, with coughing and sneezing, spreads to give aching joints and muscles, and produces a fever that makes you feel awful; but usually it has gone in 5–10 days and most people make a full recovery. The first answer here is the biological definition and, in the Open University course SK320, diseases are defined according to the infectious agent which produces them. This is because different infections can produce the same symptoms, and the same infectious agent can produce quite different symptoms in different people, depending on their age, genetic make-up or the tissue of the body that becomes infected. Here a distinction is made between the infectious disease caused by a particular agent and the disease symptoms. Unfortunately there is a lot of confusion in common parlance about different diseases. Often, people say that they have ‘a bit of flu’ when they have an infection with some other virus, or a bacterium that produces flu-like symptoms. Such loose terminology is understandable, since most people are firstly concerned with the symptoms of their disease. But to treat and control disease requires accurate identification of the causative agent, so this is the starting point for considering any infectious disease. Attributing cause to a disease The difficulties encountered in assigning a particular pathogen to a disease are well-illustrated by influenza. During the influenza pandemic that occurred in 1890, the microbiologist Pfeiffer isolated a novel bacterium from the lungs of people who had died of flu. The bacterium was named Haemophilus influenzae and since it was the only bacterium that could be regularly cultivated from these individuals at autopsy, it was assumed that H. influenzae was the causative agent of flu. Again, in the 1918 flu pandemic, the bacterium could be regularly cultivated from people who had died of flu with pneumonia. So it was thought that flu was caused by the bacterium, and H. influenzae came to be called the ‘influenza bacillus’. The role of H. influenzae was only brought into question in the early 1930s, when Smith, Andrews and Laidlaw showed that it was possible to transfer a flu-like illness from the nasal washings of an infected person to ferrets, using a bacteria-free filtrate. These studies demonstrated that the pathogen was in fact much smaller than any known bacterium and paved the way to the identification of influenza viruses (Figure 1). Figure 1 Influenza viruses are small and use RNA as their genetic material. They have irregular shapes and the outer envelope (the dark-striped outer layer of each virus in this electron micrograph), which is derived from the plasma membrane of the host cell, contains viral proteins. Why do you suppose that H. influenzae was incorrectly identified as the causative agent of flu? Reveal answer The bacterium fulfils two of Koch’s postulates: it is regularly found in serious flu infections and it can be cultured in pure form on artificial media. Moreover, at that time no-one knew what a virus was, and everyone was thinking in terms of bacterial causes for infectious diseases. Although the precise role of H. influenzae in the 1890 and 1918 flu pandemics is not clear, it is likely that the bacteria were present and acting in concert with the flu virus to produce the pneumonia experienced. Such synergy between virus and bacteria was demonstrated by Shope in 1931. He infected pigs with a bacterial-free filtrate (containing swine influenza virus) with or without the bacteria, and showed that the disease produced by the bacteria and filtrate together was more severe than that produced by either one alone (Van Epps, 2006). In its role of co-pathogen, H. influenzae is only one of a number of bacteria that can exacerbate the viral infection. This highlights a very important point. In the tidy world of a microbiology or immunology laboratory, scientists typically examine the effect of one infectious agent in producing disease. In the real world, people often become infected with more than one pathogen. Indeed, infection with one agent often lays a person open to infection with another, as immune defences become overwhelmed. For this reason, a particular disease as seen by physicians may be due to a combination of pathogens. 1.2 Influenza infection in humans Influenza is an acute viral disease that affects the respiratory tract in humans. The virus is spread readily in aerosol droplets produced by coughing and sneezing, which are symptoms of the illness. Other symptoms include fatigue, muscle and joint pains and fever. Following infection, the influenza virus replicates in the cells lining the host’s upper and lower respiratory tract. Virus production peaks 1–2 days later, and virus particles are shed in secretions over the following 3–4 days. During this period, the patient is infectious and the symptoms are typically at their most severe. After one week, virus is no longer produced, although it is possible to detect viral antigens for up to 2 weeks. Immune responses are initiated immediately after the virus starts to replicate, and antibodies against the virus start to appear in the blood at 3–4 days post infection. These continue to increase over the following days and persist in the blood for many months. In a typical flu infection, the virus is completely eliminated from the host’s system within 2 weeks. This is sterile immunity: the virus cannot be obtained from the patient after recovery from the disease. Figure 2 Time course of a typical flu infection Production of the virus (purple bar) starts early after infection and is maximal within two days. The infection is contained during this period by various immune defences. Specific antibody production starts to appear by day 3 (yellow bar), and this contributes to the elimination of the virus. Symptoms (red bar) coincide with the production of the virus. Long description For infants, older people, and those with other underlying diseases (e.g. of the heart or respiratory system) an infection with flu may prove fatal. However, the severity of a flu epidemic and the case fatality rate depend on the strain of flu involved and the level of immunity in the host population. During a severe epidemic, there are typically thousands more deaths than would normally be expected for that time of year, and these can be attributed to the disease. Although older people are usually most at risk from fatal disease, this is not always so. In the 1918 flu pandemic there was a surprisingly high death rate in people aged 20–40 and this was also the case for the 2009 ‘swine flu’ pandemic. Figure 3 Mortality according to age in the 1918 USA flu epidemic: 1917 (red) and 1918 (blue) rates in males and females. This epidemic was notable because it particularly affected people aged 20–40 year Older people are often most severely affected during infectious disease outbreaks because they may have a less effective immune response than younger people, or a reduced capacity to repair and regenerate tissue damaged by the infection. However, there are circumstances where older people may be more resistant to infection than younger people because they may have already encountered the disease (in their youth) and could retain some immunity and so be less susceptible than younger people who have not encountered the disease before. 1.3 Influenza infection in other species Influenza viruses infect a wide range of species, including pigs, horses, ducks, chickens and seals. In most of these other species the virus produces an acute infection. For example, in most of the mammals the symptoms are very similar to those in humans: an acute infection of the respiratory tract, which is controlled by the immune response although fatal infections occur in some species. However, in wild ducks and other aquatic birds the virus primarily infects the gut and the birds do not appear to have any physical symptoms. Despite this, ducks may remain infected for 2–4 weeks and during this time they shed virus in their faeces. Potentially this is a very important reservoir of infection; although flu viruses do not often cross the species barrier, the pool of viruses present in other species is an important genetic reservoir for the generation of new flu viruses that do infect humans. This reservoir becomes particularly important in certain farming communities or in crowded conditions where animals (especially pigs and ducks) are continuously in close proximity with humans (Branswell, 2010). Although such conditions occur in many agricultural communities throughout the world, they are typically observed in South-East and East Asia thereby contributing to these geographical areas often being the source of radically new ‘hybrid’ strains of influenza that incorporate genes from different species-specific strains. (The genetics of influenza are discussed in Section 2.3.) When strategies for controlling a disease are considered, awareness of the possible presence of an animal reservoir of infection is very important. For example, an immunisation programme against flu would substantially reduce the incidence of the current strain in humans but, because there is always a reservoir of these viruses in other animals, and these viruses are constantly mutating, another strain would inevitably emerge and be unaffected by immunisation. It is useful to distinguish diseases such as rabies, which primarily affect other vertebrates and occasionally infect humans (zoonoses), from diseases such as flu where different strains of the virus can affect several species including humans. Identify a fundamental difference between the way that zoonoses (e.g. rabies) are transmitted, and the way in which flu is transmitted. Reveal answer Flu can be transmitted from one human being to another, whereas most zoonoses, including rabies, are not transmitted between people. 2 Influenza viruses Viruses have very diverse genomes. Whereas the genomes of bacteria, plants and animals are of double-stranded DNA, the genomes of viruses can be constituted from either DNA or RNA and may be double- or single-stranded molecules. Usually, DNA is a double-stranded molecule with paired, complementary strands (dsDNA) and RNA is a single stranded molecule (ssRNA). However, some viruses have single-stranded DNA genomes (ssDNA) and some have double-stranded RNA genomes (dsRNA). The type of nucleic acid found in the genome depends on the group of viruses involved. RNA encodes protein in all living things, and the sequence of bases in the RNA determines the sequence of amino acids in the protein. A strand of RNA which has the potential to encode protein is said to be ‘positive sense’ (+). If a strand of RNA is complementary to this, then it is ‘negative sense’ (–). Negative-sense RNA must first be copied to a complementary positive-sense strand of RNA before it can be translated into protein. The description of the influenza genome as negative-sense ssRNA means that its RNA cannot be translated without copying first. This copying is performed by influenza’s viral RNA polymerase, a small amount of which is packaged with the virus, ready to begin copying the viral genome once it enters a host cell. Viral RNA polymerase consists of three subunits: PB1, PB2, and PA, encoded separately by the first three viral RNA strands. Understanding the way in which different viruses replicate is important, since it allows the identification of particular points in their life-cycle that may be susceptible to treatment with antiviral drugs. Classification Viruses are classified into different families, groups and subgroups in much the same way as are species of animals or plants. As you have already read, the influenza viruses are (–)ssRNA organisms (Baltimore group V) and belong to a family called the Orthomyxoviruses . They fall into three groups: influenza A, B and C. Type A viruses are able to infect a wide variety of endothermic (warm-blooded) animals, including mammals and birds, and analysis of their viral genome indicates that all strains of influenza A originated from aquatic birds. By contrast, types B and C are mostly confined to humans. At any one time, a number of different strains of virus may be circulating in the human population. Families, groups and strains of virus Viruses were originally classified into different groups according to similarities in their structure, mode of replication and disease symptoms. For example, the Orthomyxoviruses include viruses that cause different types of influenza, while Paramyxoviruses include the viruses that cause measles and mumps. Such large groupings are often called a family of viruses. The families can be subdivided into smaller groups, such as influenza A, B and C. Even within a single such group of viruses there can be an enormous level of genetic diversity, and this is the basis of the different strains. As an example, two HIV particles from the same individual may be 4% different in their genome; compare this with the 1% difference between the genomes of humans and chimpanzees, which are different species. 2.1 Structure of influenza The structure of influenza A is shown schematically in Figure 4. The viral genomic RNA, which consists of 8 separate strands , is enclosed by its associated nucleoproteins to make a ribonucleoprotein complex (RNP), and this is contained in the central core of the virus (the capsid). The nucleoproteins are required for viral replication and packing of the genome into the new capsid, which is formed by M1-protein (or matrix protein). The M1-protein is the most abundant component of the virus, constituting about 40% of the viral mass; it is essential for the structural integrity of the virus and to control assembly of the virus. Figure 4 Structure of an influenza A virus. The capsid, formed by M1-protein, also contains the viral genome and a number of enzymes required for viral replication. The viral envelope is a lipid bilayer formed from the plasma membrane of the host cell, which contains two virus-encoded proteins, haemagglutinin and neuraminidase. Orthomyxoviruses have a capsid surrounded by a phospholipid bilayer derived from the plasma membrane of the cell that produced the virus. This layer is the virus’s envelope. Two proteins, haemagglutinin and neuraminidase, are found on the viral envelope. These proteins are encoded by the viral HA and NA genes respectively and are inserted into the plasma membrane of the infected cell before the newly-produced viruses bud off from the cell surface. The haemagglutinin can bind to glycophorin, a type of polysaccharide that contains sialic acid residues, and which is present on the surface of a variety of host cells. The virus uses the haemagglutinin to attach to the host cells that it will infect. Antibodies and drugs against haemagglutinin are therefore particularly important in limiting the spread of the virus, since they prevent it from attaching to new host cells. Neuraminidase is an enzyme that cleaves sialic acid residues from polysaccharides. It has a role in clearing a path to the surface of the target cell before infection, namely, digesting the components of mucus surrounding epithelial cells in the respiratory system. Similarly, neuraminidase also promotes release of the budding virus from the cell surface after infection. The structures of influenza B and influenza C are broadly similar to that of Type A, although in influenza C the functions of the haemagglutinin and the neuraminidase are combined in a single molecule, haemagglutinin esterase. This molecule binds and cleaves a less common type of sialic acid. Influenza C does not normally cause clinical disease or epidemics, so the following discussion is confined to influenza A and B. 2.2 Designation of strains of influenza A considerable number of genetically different strains of influenza A have been identified, and these are classified according to where they were first isolated and according to the type of haemagglutinin and neuraminidase they express. For example ‘A/Shandong/9/93(H3N2)’ is an influenza A virus isolated in the Shandong province of China in 1993 – the ninth isolate in that year – and it has haemagglutinin type 3 and neuraminidase type 2. At the start of the twenty-first century, the major circulating influenza A strains are H1N1 (‘swine flu’) and H3N2. At least 16 major variants of haemagglutinin and 9 variants of neuraminidase have been recognised, but to date most of these have only been found in birds. The designation for influenza B is similar, but omits the information on the surface molecules, for example: ‘B/Panama/45/90’. As you will see later, accurate identification of different strains of flu is crucial if we are to control epidemics by vaccination programmes. 2.3 Genomic diversity of influenza The genome of flu viruses consists of around 14 000 nucleotides of negative-sense single-stranded RNA. Compare this number to the approximately 3 billion nucleotides found in the human genome or the 150 billion nucleotides of the genome of the marbled lungfish (the largest genome known in vertebrates). The genome of influenza viruses is segmented, into 8 distinct fragments of RNA containing 11 genes and encoding approximately 14 proteins (see Table 1 below). This structure has significance for the spread of the virus and the severity of disease symptoms. Cases of influenza generally arise in two main ways: by provoking seasonal annual outbreaks or epidemics and, less commonly, through global pandemics. As you will see shortly, both of these phenomena occur as consequences of the fact that the virus uses RNA as its genetic template and that this RNA genome is segmented into discrete strands. Table 1 The genome of influenza virus. Note that a single RNA segment may encode for more than one protein due to alternative reading frames. Gene name RNA strand (segment number) Function(s) of protein encoded by this gene PB2 (polymerase basic 2) 1 A subunit of viral RNA polymerase involved in cleaving the cap structure of host cell mRNA and generating primers that are subverted for use in the synthesis of viral RNA. PB1 (polymerase basic 1) 2 Core subunit of viral RNA polymerase. Required for polymerase assembly. PB1-F2 2 Binds to components of the host mitochondria, sensitising the cell to apoptosis and contributing to pathogenicity. PA (polymerase acidic) 3 A subunit of viral RNA polymerase which also has protease activity of unknown function. HA (haemagglutinin) 4 Antigenic glycoprotein used for binding to (infecting) the host cell. NP (nucleoprotein) 5 RNase resistant protein. Binds viral genomic RNA to form stable ribonucleoproteins and targets these for export from the host nucleus into the cytosol. Also involved in viral genome packaging and viral assembly. NA (neuraminidase) 6 Cleaves sialic acid. Important for releasing viral particles from host cell. M1 (matrix 1) 7 Binds viral genomic RNA and forms a coat inside the viral envelope in virions. Inside the host cell, it starts forming a layer under patches of the membrane rich in viral HA, NA, and M2 and so facilitates viral assembly and budding from the host cell. M2 (matrix 2) 7 Transmembrane ion channel protein. Allows protons into the virus capsid, acidifying the interior, destabilising binding of M1 to the viral genomic RNA which leads to uncoating of the viral particle inside the host cell. NS1 (non-structural 1) 8 Inhibits nuclear export of the host’s own mRNA, thereby giving preference to viral genomic RNA. Blocks the expression of some host inflammatory mediators (interferons) and interferes with T cell activation*. NS2/NEP (non-structural 2/ nuclear export protein) 8 Mediates the export of viral genomic RNA from the host nucleus to the cytoplasm. * Interferons and T cells are involved in the immune response to pathogens. The influenza virus is a successful pathogen because it is constantly changing. How might having a segmented genome promote the evolution of new strains of influenza virus? answer: If a cell is infected with more than one strain of virus at the same time, then a new strain can be generated simply by mixing RNA strands from different viruses. 2.3.1 Creation of new viral strains Part of the success of influenza as a pathogen is because segmented genome improves the virus’s potential to evolve into new strains through the combination of different RNA stands. This mixing of the genetic material from different viral strains to produce a new strain is termed genetic reassortment. For instance, the virus that caused the 2009 H1N1 ‘swine flu’ pandemic comprises a quadruple reassortment of RNA strands from two swine virus, one avian virus, and one human influenza virus: the surface HA and NA proteins derive from two different swine influenzas (H1 from a North American swine influenza and N1 from a European swine influenza) the three components of the RNA polymerase derive from avian and human influenzas (PA and PB2 from the avian source, PB1 from the human 1993 H3N2 strain) the remaining internal proteins derive from the two swine influenzas (MacKenzie, 2009). This does not necessarily mean that all four viruses infected the same animal at once. The new strain was likely the result of a reassortment of two swine influenza viruses, one from North America and one from Europe. The North American virus may itself have been the product of previous reassortments, containing a human PB1 gene since 1993 and an avian PA and PB2 genes since 2001. The presence of avian influenza RNA polymerase genes in this virus was especially worrying, since the avian polymerase is thought to be more efficient than human or swine versions, allowing the virus to replicate faster and thus making it more virulent. Similar avian RNA polymerase genes are what make H5N1 bird flu extremely virulent in mammals and what made the 1918 human pandemic virus so lethal in people. This mixing of genes from two or more viruses (whether from the same host species or from different species) can cause major changes in the antigenic surface proteins of a virus, such that it is no longer recognised by the host’s immune system. This antigenic shift is described in more detail in Section 3 (specifically, Box 2). In contrast to the major genetic changes caused by reassortment, influenza viruses also undergo constant, gradual, genetic changes due to errors made by their RNA polymerases. 2.4 Infection and replication Influenza RNA polymerase lacks the ability to recognise and repair any errors that occur during genome duplication, resulting in mistakes in copying its viral RNA about once in every 10 000 nucleotides. Because the influenza genome only contains approximately 14 000 nucleotides, this means that, on average, each new virus produced differs by 1 or 2 nucleotides from its ‘parent’. The slow accumulation of random genetic changes, especially in the antigenic surface proteins, explains why antibodies that were effective against the virus one year may be less effective against it in subsequent years. This gradual change in the nature of viral antigens is known as antigenic drift. The replication cycle of influenza is illustrated in Figure 5 Figure 5 Replication cycle of a flu virus. Long description Influenza is spread in aerosol droplets that contain virus particles (or by desiccated viral nuclei droplets), and infection may occur if these come into contact with the respiratory tract. Viral neuraminidase cleaves polysaccharides in the protective mucus coating the tract, which allows the virus to reach the surface of the respiratory epithelium. The haemagglutinin now attaches to glycophorins (sialic-acid-containing glycoproteins) on the surface of the host cell, and the virus is taken up by endocytosis into a phagosome. Acidic lysosomes fuse with the phagosome to form a phagolysosome and the pH inside the phagolysosome falls. This promotes fusion of the viral envelope with the membrane of the phagolysosome, triggering uncoating of the viral capsid and release of viral RNA and nucleoproteins into the cytosol. The viral genomic RNA then migrates to the nucleus where replication of the viral genome and transcription of viral mRNA occur. These processes require both host and viral enzymes. The viral negative-stranded RNA is replicated by the viral RNA-dependent RNA polymerase, into a positive-sense complementary RNA (cRNA), and these positive and negative RNA strands associate to form double-stranded RNA (dsRNA). The cRNA strand is subsequently replicated again to produce new viral genomic negative-stranded RNA. Some of the cRNA is also processed into mRNA for translation of viral proteins. The infection cycle is rapid and viral molecules can be detected inside the host cell within an hour of the initial infection. The envelope glycoproteins (haemagglutinin and neuraminidase) are translated in the endoplasmic reticulum, processed and transported to the cell’s plasma membrane. The viral capsid is assembled within the nucleus of the infected cell. The capsid moves to the plasma membrane, where it buds off, taking a segment of membrane containing the haemagglutinin and neuraminidase, and this forms the new viral envelope. Influenza virus budding from the surface of an infected cell is shown in Figure 6. Figure 6 Flu virus particles budding from the surface of an infected cell. Long description From the description above, identify a process or element in the replication cycle which is characteristic of the virus, and which would not normally occur in a mammalian cell. Answer: The replication of RNA on an RNA template with the production of double-stranded RNA would never normally occur in a mammalian cell. Double stranded RNA is therefore a signature of a viral infection. Significantly, cells have a way of detecting the presence of dsRNA, and this activates interferons: molecules involved in limiting viral replication. 2.5 Cellular pathology of influenza infection Flu viruses can infect a number of different cell types from different species. This phenomenon is partly because the cellular glycoproteins which are recognised by viral haemagglutinin are widely distributed in the infectious agent. What is the term for the property of viruses that allows them to only replicate in particular cell types? Reveal answer A second reason why the virus can infect a variety of cell types is that the replication strategy of flu is relatively simple: ‘infect the cell, replicate as quickly as possible and then get out again’. This is the cytopathic effect of the virus. Cell death caused directly by the virus can be distinguished from cell death caused by the actions of the immune system as it eliminates infected cells. The effects of cell death Cell death impairs the function of an infected organ and often induces inflammation, a process that brings white cells (leukocytes) and molecules of the immune system to the site of infection. In the first instance, the leukocytes are involved in limiting the spread of infection; later they become involved in combating the infection, and in the final phase they clear cellular debris so that the tissue can repair or regenerate. The symptoms of flu experienced by an infected person are partly due to the cytopathic effect of the virus, partly due to inflammation and partly a result of the innate immune response against the virus. The severity of the disease largely depends on the rate at which these processes occur. In most instances, the immune response develops sufficiently quickly to control the infection and patients recover. If viral replication and damage outstrip the development of the immune response then a fatal infection can occur. In severe flu infections, the lungs may fill with fluid as the epithelium lining the alveoli (air sacs) is damaged by the virus. The fluid is ideal for the growth of bacteria, and this can lead to a bacterial pneumonia, in which the lungs become infected with one or more types of bacteria such as Haemophilus influenzae. Damage to cells lining blood vessels can cause local bleeding into the tissues, and this form of ‘fulminating disease’ was regularly seen in post-mortem lung tissues of people who died in the 1918 pandemic. 3 Patterns of disease In humans, pigs and horses, flu viruses circulate through populations at regular intervals. The disease is endemic in tropical regions for all of these host groups (i.e. it is continually present in the community). In temperate latitudes, infections are usually seasonal or epidemic, with the greatest numbers occurring in the winter months (Figure 7). Epidemics also occur sporadically in sea mammals and poultry, and in these species high mortality is typical. Figure 7 Epidemic patterns of flu in temperate latitudes. The graph shows notifications of flu in the USA each week from 1994–1997. Long description In most years, flu in humans affects a minority of the population, the disease course is not very severe and the level of mortality is not great. In such years the influenza virus is slightly different from the previous year due to antigenic drift, which results in the accumulation of genetic mutations that cause the molecules present on the surface of the virus change progressively. In this scenario, the virus is not significantly different from the previous year so that the host’s immune system can more easily mount an effective response than it could to a completely new strain. However, at irregular intervals the virus undergoes an antigenic shift. This process only occurs in influenza A viruses, typically every 10–30 years, and it is associated with severe pandemics, serious disease and high mortality (see Box 2). In Section 2.3 you read that strains of influenza are differentiated and designated using a simple system of numbers and letters that depend on their surface antigens. More commonly, however, strains responsible for pandemics are often given a common name according to the area in the world from which they were thought to originate, or the species they mainly affected before becoming transferred to humans (see Table 2). Evidence suggests, however, that in the twentieth century the major flu pandemics all originated in China, with the exception of the 1918 pandemic, which first occurred in the USA. Major flu pandemic strains of the twentieth century. Year Designation Common name 1900 H3N8 (none) 1918 H1N1 Spanish flu西班牙 1957 H2N2 Asian flu亞洲 1968 H3N2 Hong Kong flu香港 1977 H1N1 Russian flu蘇聯 1997 H5N1 Avian flu禽 The rise of H5N1 – an example of antigenic shift In 1997, a new strain of influenza A, H5N1, was identified in Hong Kong. The strain was rife in chickens and a few hundred people had become infected. Mortality in these individuals was very high, (6 of 18 died), and so there was serious concern that it marked the beginning of a new pandemic. The authorities in Hong Kong responded by a mass cull of poultry in the region and about 1.5 million chickens were slaughtered. H5N1 did not spread easily from person to person and no further cases were reported in people following the slaughter. Whether the H5N1 outbreak was an isolated incident of a strain spreading from chicken to humans, or whether it was the start of a major pandemic which was nipped in the bud, cannot be known. Subsequent analysis showed that the high virulence of the new strain could partly be related to the new variant of haemagglutinin (H5), and partly to a more efficient viral polymerase. This outbreak clearly demonstrates the way in which bird influenza can act as a source of new viral strains, and shows that such new strains may be very dangerous to humans. Since the discovery of the influenza virus in the 1930s it has been possible to isolate and accurately identify each of the epidemic strains, but, as earlier strains of virus have now died out, it has been necessary to infer their identity by examining the antibodies in the serum of affected people. Antibodies and the ability of the immune system to respond to a strain of flu are much more persistent than the virus itself. It is thus possible to analyse antibodies to determine which types of haemagglutinin and neuraminidase they recognise long after the virus itself has gone. One can then deduce which type of influenza virus that person contracted earlier in their life (as explained in Section 5.2). 3.1 Tracking the emergence of new strains Influenza is one of several diseases monitored by the WHO Global Alert and Response   (GAR) network (WHO, 2011a), comprising 110 ‘sentinel’ laboratories in 82 countries. The organisation’s surveillance and monitoring of the disease then forms part of their Global Influenza Programme (GIP), and they use data gathered from participating countries to: provide countries, areas and territories with information about influenza transmission in other parts of the world to allow national policy makers to better prepare for upcoming seasons provide data for decision making regarding recommendations for vaccination and treatment describe critical features of influenza epidemiology including risk groups, transmission characteristics, and impact monitor global trends in influenza transmission inform the selection of influenza strains for vaccine production (WHO, 2011b). The influenza data from the sentinel laboratories is fed into a global surveillance programme, started by the WHO in 1996, called FluNet (WHO, 2011c), which is one of the tools that facilitates the actions described above. Typically a flu vaccine contains material from the main influenza A strains and an influenza B strain, so that an immune response is induced against the most likely infections. Usually the scientists predict correctly and immunised people are effectively protected against the current strains (>90% protection). However, the prediction is occasionally incorrect, or a new strain develops during the time that the vaccine is being manufactured. In this case the vaccine generally provides poor protection. What can you deduce about immunity against flu infection from the observations on vaccination above? answer The immune response is strain-specific. If you are immunised against the wrong strain of flu, then the response is much less effective and you are more likely to contract the disease. 3.2 Immune responses to influenza The immune system uses different types of immune defence against different types of pathogen. The responses against flu are typical of those which are mounted against an acute viral infection, but different from the responses against infection by bacteria, worms, fungi or protist parasites. When confronted with an acute viral infection, the immune system has two major challenges: The virus replicates very rapidly, killing the cells it infects. Since a specific immune response takes several days to develop, the body must limit the spread of the virus until the immune defences can come into play. Viruses replicate inside cells of the body, but they spread throughout the host in the blood and tissue fluids. Therefore, the immune defences must recognise infected cells (intracellular virus) and destroy them. But the immune system must also recognise and eradicate free virus in the tissue fluids (extracellular virus) in order to prevent the virus from infecting new cells.. 3.2.1 Summary of the response How does the body act quickly to limit viral spread? When a virus infects a cell of the body, the molecular machinery for protein synthesis within the cell is usurped as the virus starts to produce its own nucleic acids and proteins. The cell detects the flu dsRNA and other viral molecules and releases interferons, which bind to receptors on neighbouring cells and cause them to synthesise antiviral proteins. If a virus infects such cells they resist viral replication, so fewer viruses are produced and viral spread is delayed. Also, in the earliest stages of a virus infection the molecules on the cell surface change. Cells lose molecules that identify them as normal ‘self’ cells. At the same time they acquire new molecules encoded by the virus. A group of large, granular lymphocytes recognise these changes and are able to kill the infected cell. This function is called ‘natural killer’ cell action and the lymphocytes that carry it out are termed NK cells. Non-adaptive and adaptive responses The actions of both interferons and NK cells in combating infection by influenza occur early in an immune response, and are not specific for the flu virus. These defences occur in response to many different kinds of viral infection, and they are part of our natural, or non-adaptive, immune responses. Note that immunologists use the term non-adaptive to indicate a type of response that does not improve or adapt with each subsequent infection. This is quite different to its use in evolutionary biology, where it means ‘not advantageous’. Such non-adaptive immune responses slow the spread of an infection so that specific, or adaptive, immune defences can come into play. The key features of an adaptive immune response are specificity and memory. The immune response is specific to a particular pathogen, and the immune system appears to ‘remember’ the infection, so that if it occurs again the immune response is much more powerful and rapid. Because an immune response is highly specific to a particular pathogen it often means that a response against one strain of virus is ineffective against another – if a virus mutates then the lymphocytes that mediate adaptive immunity are unable to recognise the new strain. There are two principal arms of the adaptive immune system, mediated by different populations of lymphocytes. One group, called T-lymphocytes, or T cells (which develop in the thymus gland, overlying the heart), recognises antigen fragments associated with cells of the body, including cells which have become infected. A set of cytotoxic T cells (Tc) specifically recognises cells which have become infected and will go on to kill them. In this sense they act in a similar way to NK cells. However they differ from NK cells in that Tc cells are specific for one antigen or infectious agent, whereas NK cells are non-specific. The second group of lymphocytes are B cells (that differentiate in the bone marrow), which synthesise antibodies that recognise intact antigens, either in body fluids or on the surface of other cells. Activated B cells progress to produce a secreted form of their own surface antibody. Antibodies that recognise the free virus act to target it for uptake and destruction by phagocytic cells. Therefore the T cells and NK cells deal with the intracellular phase of the viral infection, while the B cells and antibodies recognise and deal with the extracellular virus. The two reactions described above are illustrated in Figure 8. Figure 8 Immune defences against flu viruses. Antibodies can block the spread of the virus by preventing them from attaching to host cells. Infected cells release interferon, which signals to neighbouring cells to induce resistance. Natural killer cells (NK) and cytotoxic T cells (Tc) recognise and kill virally-infected cells. Long description You might ask why it takes the adaptive immune response so long to get going. The answer is that the number of T cells and B cells that recognise any specific pathogen is relatively small, so first the lymphocytes which specifically recognise the virus must divide so that there are sufficient to mount an effective immune response. This mechanism is fundamental to all adaptive immune responses. How do pandemic strains of influenza A come about? Answer Question 2 Pandemic strains of influenza A normally arise by simultaneous infection of a non-human host (typically poultry or pigs) with two or more strains of influenza A. Reassortment of the eight viral segments from each virus allows the generation of a new hybrid virus type, with a completely novel surface structure that has never been seen before by a host immune system (antigenic shift). 4 Antiviral treatments Two classes of antiviral drugs are used to combat influenza: neuraminidase inhibitors and M2 protein inhibitors. Why are antibiotics not used to combat influenza? Reveal answer Neuraminidase inhibitors neuraminidase is an enzyme that is present on the virus envelope and cleaves sialic acid groups found in the polysaccharide coating of many cells (especially the mucus coating of the respiratory tract). Neuraminidase is used to clear a path for the virus to a host cell and facilitates the shedding of virions from an infected cell. Inhibition of neuraminidase therefore helps prevent the spread of virus within a host and its shedding to infect other hosts. The two main neuraminidase inhibitors currently in clinical use are zanamivir (trade name Relenza) and oseltamivir (trade name Tamiflu). These are effective against influenza A and B, but not influenza C which exhibits a different type of neuraminidase activity that only cleaves 9-O-acetylated sialic acid. M2 inhibitors Recall from Table 1 that the influenza M2 protein forms a pore that allows protons into the capsid, acidifying the interior and facilitating uncoating. Drugs such as amantadine (trade name Symmetrel) and rimantadine (trade name Flumadine) block this pore, preventing uncoating and infection. However, their indiscriminate use in ‘over-the-counter’ cold remedies and farmed poultry has allowed many strains of influenza to develop resistance. Influenza B has a different type of M2 protein which is largely unaffected by these drugs. 5 Diagnosis of influenza Many diseases produce symptoms similar to those of influenza; in fact, ‘flu-like’ is a term that is frequently used to describe several different illnesses. Since influenza spreads rapidly by airborne transmission and is a life-threatening condition in certain vulnerable groups, it is important that cases of the disease are identified as quickly as possible, so that preventative measures may be taken. Most viral infections are not treated, although antiviral drugs such as zanamivir are used for potentially life-threatening cases or where the risk of transmission is high (as occurs during a pandemic). Which sites in the body should be sampled for diagnosis? answer The influenza virus infects the respiratory tract and is spread by coughing and sneezing, so specimens should be taken from the nose, throat or trachea. In practice, the best specimens are nasal aspirates or washes, but swabs of the nose or throat may be used if they are taken vigorously enough to obtain cells. Ideally, samples should be taken within three days of the onset of illness, and all specimens need to be preserved in a transport medium and kept chilled until they reach the clinical microbiology laboratory. 5.1 Initial identification of influenza infection Oral swabs or nasal aspirates are initially screened for the presence of a variety of respiratory viruses. This is done by extracting RNA from the sample and subjecting it to a reverse-transcription polymerase chain reaction (RT-PCR) as described below: Initially the RNA sample is reverse transcribed into complementary DNA (cDNA), using a commercially-available reverse transcriptase enzyme. The cDNA is then used in a standard PCR reaction to detect and amplify a short sequence of nucleotides specific to the virus. Multiple DNA sequences, each specific for a different type of virus, can be amplified in the same reaction, provided that these sequences are of different lengths. Each of the different amplified sequences is separated from the others when the entire sample is subjected to gel electrophoresis (an analytical technique in which molecules of different sizes move at various rates through a gel support in an applied electric field, thus making it possible to identify specific molecules.) PCR technique. Typically, nucleotide sequences specific to 5 types of virus are searched for in each sample: influenza A, influenza B, respiratory syncytial virus (Baltimore group V, (–)ssRNA virus, and a major cause of respiratory illness in young children), adenoviruses and enteroviruses. Those samples that test positive for influenza in the RT-PCR reaction are inoculated into cells in culture. Sufficient virus for a limited number of tests can be produced from such cultures within 24 hours, but they are often maintained for up to a week. 5.2 Determining the subtype of influenza Immunofluorescence Confirmation of a case of influenza is usually achieved by performing tests on some of the inoculated cultured cells using reference fluorescent-labelled antisera provided by the WHO. A reference antiserum is a sample serum known to contain antibodies specific for the molecule to be assayed (in this case, haemagglutinin or neuraminidase). These antisera are prepared using purified haemagglutinin and neuraminidase and are monospecific, each antibody reacting only with one epitope e.g. H1 or H3. For the test, an antibody is added to a sample of inoculated cells. Following a wash step, if the antibody remains bound to the cells then they fluoresce under appropriate illumination, indicating the presence of viral antigen on the cell surface. This diagnostic technique can identify influenza virus on infected cells in as little as 15 minutes. A positive result not only confirms the RT-PCR data, but gives additional information on the subtype of the virus. Further PCR analyses Standardised RT-PCR protocols exist to look for the presence of different haemagglutinin and neuraminidase subtypes, chiefly H1, H3, H5, N1 and N2. (Poddar, 2002). If the PCR analysis indicates a dangerous strain of influenza A e.g. H5N1, then it is instantly sent to a WHO reference laboratory for further tests. Haemagglutination assays Influenza has haemagglutinins protruding from its viral envelope, which it uses to attach to host cells prior to entry. These substances form the basis of a haemagglutination assay, in which viral haemagglutinins bind and cross-link (agglutinate) red blood cells added to a test well, causing them to sink to the bottom of the solution as a mat of cells. If agglutination does not occur, then the red blood cells are instead free to roll down the curved sides of the tube to form a tight pellet. A related test called a haemagglutination-inhibition assay (HAI), incorporates antibodies against different subtypes of viral haemagglutinin. The antibodies bind and mask the viral haemagglutinin, preventing it from attaching to and cross-linking red blood cells. A HAI assay can be set up in one of two ways: either a known reference antibody is added to an unknown virus sample, or known reference viral haemagglutinin is added to a sample of patient serum containing antibodies against influenza. This second version of the HAI assay can therefore be used long after the infection has passed, when virions are no longer present. Haemagglutination and HAI assays have the advantage that they are simple to perform and require relatively cheap equipment and reagents. However, they can be prone to false positive or false negative results, if the sample contains non-specific inhibitors of haemagglutination (preventing agglutination) or naturally occurring agglutinins of red blood cells (causing agglutination). If a confirmed influenza A isolate reacts weakly or not at all in HAI then this indicates an unknown variant of influenza A and the sample is immediately sent to a WHO reference laboratory for further tests. Neuraminidase inhibition assay Typing influenza isolates in terms of their neuraminidase makes use of the enzyme activity of this glycoprotein. The neuraminidase inhibition assay is performed in two parts. The first part determines the amount of neuraminidase activity in a patient influenza sample, as outlined in Figure 9a. A substrate (called fetuin) that is rich in sialic acid residues is added to a sample of the influenza virus, and the viral neuraminidase enzyme cleaves the substrate to produce free sialic acid. Addition of a substance that inactivates the neuraminidase stops the reaction, and a chromogen (a colourless compound that reacts to produce a coloured end-product) that turns pink in the presence of free sialic acid is added. The intensity of the pink colour is proportional to the amount of free sialic acid and can be measured using a spectrophotometer. This assay of neuraminidase activity allows the appropriate amount of virus sample to be determined, and this quantity is then used in the second part of the assay. If too much or too little virus is used, the resulting changes, and therefore the neuraminidase, may be undetectable. In the second part of the assay (Figure 9b), viral samples from the patient are incubated with anti-neuraminidase reference antisera. Each of the reference antisera used for this test has antibodies that bind one particular neuraminidase variant, e.g. N1 or N2. How can these antisera be used to type the neuraminidase variant? Figure 9 The neuraminidase inhibition assay. (a) Assay of neuraminidase activity. (b) The inhibition of the assay itself. 6 Summary of the unit . A single pathogen can produce different types of disease in different people. Genetic variation in a pathogen can also affect the type of disease it produces. To understand this we need to know something of the genetic and social differences in the host population, and of the diversity of the pathogen. The symptoms of a particular disease may be produced by different pathogens or by a combination of pathogens. To understand this requires some knowledge of pathology and cell biology. Some diseases, such as flu, affect humans and several other animal species, whereas others are more selective in their host range. The basic biology of different pathogens underlies these differences. Flu is a disease that can be contracted several times during a lifetime, but many other infectious diseases are only ever contracted once. To understand this we need to look at how the immune system reacts to different pathogens, and how responses vary depending on the pathogen. Outbreaks of flu occur regularly, but some epidemics are much more serious than others. This requires an understanding of aspects of virology, immunology, evolutionary biology and epidemiology. In 1892, German bacteriologist Richard Pfeiffer isolated what he thought was the causative agent of influenza. The culprit, according to Pfeiffer, was a small rod-shaped bacterium that he isolated from the noses of flu-infected patients (1). He dubbed it Bacillus influenzae (or Pfeiffer's bacillus). Few doubted the validity of this discovery, in large part because bacteria had been shown to cause other human diseases, including anthrax, cholera, and plague. Figure 1 Richard Shope, 1936. The filtration question When history's deadliest influenza pandemic began in 1918, most scientists believed that Pfeiffer's bacillus caused influenza. With the lethality of this outbreak (which killed an estimated 20 to 100 million worldwide) came urgency—researchers around the world began to search for Pfeiffer's bacillus in patients, hoping to develop antisera and vaccines that would protect against infection. In many patients, but not all, the bacteria were found. Failures to isolate B. influenzae (now known as Haemophilus influenzae) were largely chalked up to inadequate technique, as the bacteria were notoriously difficult to culture (2). The first potential blow to Pfeiffer's theory came from Peter Olitsky and Frederick Gates at The Rockefeller Institute. Olitsky and Gates took nasal secretions from patients infected with the 1918 flu and passed them through Berkefeld filters, which exclude bacteria. The infectious agent—which caused lung disease in rabits—passed through the filter, suggesting that it was not a bacterium (3, 4). Although the duo had perhaps isolated the influenza virus (which they nevertheless referred to as an atypical bacterium called Bacterium pneumosintes), other researchers could not reproduce their results. One of the doubters was Oswald Avery (Rockefeller Institute), who developed a culture media—chocolate agar—that optimized the growing conditions for B. influenzae and thus minimized false negative results from patient samples. Thus, the idea that flu was transmitted by a filterable agent (or virus) was dismissed. Insights from pigs Olitsky and Gates would not be vindicated until a decade later, when Shope—a young physician from Iowa then working on hog cholera at the Rockefeller Institute—turned his attention to swine influenza. Pig farmers in Iowa had reported two outbreaks—one in 1918 and another in 1929—of a highly contagious, influenza-like disease among their animals. The disease bore such a remarkable resemblance to human flu that it was named swine influenza. Shope and his mentor Paul Lewis took mucus and lung samples from the infected pigs and attempted to isolate the disease-causing agent. They quickly isolated a bacterium that looked exactly like Pfeiffer's human bacterium (and was thus called B. influenzae suis), but when they injected the bacteria into pigs, it caused no disease (5). Shope then filtered the samples and, like Olitsky and Gates, found that the filtrate contained the infectious agent. Shope's filtrate caused a highly contagious, influenza-like disease in pigs—albeit a more mild one than seen in naturally-infected pigs. Mixing the filtrate with the bacterium reproduced the severe disease. He concluded—correctly—that the filterable agent caused the infection, which then facilitated secondary infection with the bacterium (6). Shope published his results in a series of papers in The Journal of Experimental Medicine (5, 6). Using Shope's technique, Wilson Smith, Christopher Andrewes, and Patrick Laidlaw (National Institute for Medical Research, UK) soon isolated the virus from humans (7), laying to rest any lingering doubts about the nature of the flu-inducing agent. Both Shope and the British trio later demonstrated that sera from humans that were infected with the 1918 flu virus could neutralize the pig virus, leading them to conclude that the swine virus was a surviving form of the 1918 human pandemic virus (8, 9). In fact, a related strain of flu still circulates among pigs today.

2012談2009之流感

2012談2009之流感 　　 流感是流感病毒引起的急性呼吸道感染。每年冬天幾乎都有一至兩波的流感疫情，估計在台灣造成約 3000 人死亡，其中九成為老人。流感病毒為 RNA 病毒，RNA 病毒相較於 DNA 病毒，本來就有較高的點突變率，約萬分之一機率，因此每年來襲的流感病毒都會與前一年流行的病毒些許不同（抗原漂變antigenic drift)，造成人們反覆的季節性流感感染。此外由於流感病毒的基因是8條分段的 RNA，所以可以在豬、禽、人間發生基因重組(antigenic shift抗原移變），造成大流行。使得新種流感病毒一直出現。A型流感病毒的感染宿主非常多元，幾乎多種哺乳類及鳥類都可被感染，其中以豬最為關鍵。豬同時具有人流感與禽流感病毒的細胞受體（2-6；2-3），可被兩種以上病毒同時感染，進而發生基因重組。因此會攜帶許多混種流感病毒。加上豬是重要經濟家畜，廣泛存在於農村與住家，與人類接觸頻繁，因此多次流感世界大流行都源自於豬，舉凡 1957, 1968, 2009 大流行都是如此。 　　2009 年 H1N1 新流感在短斷數個月內席捲全球，仔細分析其基因，還是北美、歐洲流感病毒在豬身上基因重組而成，令人跌破眼鏡的是此病毒的血球凝集素 (HA) 基因竟然來自 1918 年的大流行病毒，豬隻自1918和人一起被H1N1感染後，病毒株在豬隻體內並未發生大變異，但人季節性流感 H1N1則是一度在1957消失，在1970年代後才又出現，一直流行於人群至今，但已和1918株不同了。60 歲以上民眾曾經曝露在1918那株流感病毒，體內已有抗體存在，所以2009豬流感不易對此族群造成重症。1958後出生者未曾遇過此株病毒，所以毫無抵抗力，因此造成再一次的大流行。這次大流行也打破了每次大流行都來自亞洲的慣例，暴露出流感的不可預測性。 　　加強動物流感監視是控制流感流行的重要一環，由於A流感病毒廣泛存在各種物種中，要根絕幾乎是不可能的。唯有減少其傳播到人身上是可行的策略。目前的疫苗有其侷限性，發展更新一代全方位流感疫苗是必走的路。最新消息，2011，七，八月已有發表全方位疫苗在動物實驗成功。接下來在人體實驗上也需密切追蹤！極有可能對流感防範造成突破性發展！

疫苗的備製

Influenza virus growth in eggs流感病毒在雞胚上的生長 10 DECEMBER 2009 Before the development of cell culture, many viruses were propagated in embryonated chicken eggs. Today this method is most commonly used for growth of influenza virus. The excellent yield of virus from chicken eggs has led to their widespread use in research laboratories and for vaccine production. In fact the vast majority of influenza vaccines – both inactivated and infectious – are produced in chicken eggs. How is influenza virus propagated in eggs? 由於流感病毒在雞胚上生長的非常好，所以雖有細胞培養，但流感病毒的研究、疫苗備製仍是使用雞胚。尿膜囊液中富含蛋白酶，可將血凝素切開，使從HA0變HA1,HA2,才具感染力！若用細胞培養方式、則必需加蛋白酶，一般是加trypsin胰蛋白酶 The illustration below shows a cutaway view of an embryonated chicken egg. The different routes of inoculation into the egg are shown, as well as the different compartments in which viruses replicate. For propagation of influenza virus, pathogen-free eggs are used 11-12 days after fertilization. The egg is placed in front of a light source to locate a non-veined area of the allantoic cavity just below the air sac. This is marked with a pencil. After all the eggs have been ‘candled’ in this way, a small nick is made in the shell at this position using a jeweler’s scribe. Next, a hole is drilled at the top of the egg with a Dremel motorized tool. If this is not done, when virus is injected, the pressure in the air sac will simply force out the inoculum.使用受精11至12天的雞胚，用光源照射找出氣囊下方，無靜脈區的尿膜囊allantoic腔,用鉛筆標記，氣囊先打小洞洩壓，方便植入病毒。用27號針將病毒種入尿膜囊allantoic腔中，在37度培養2天。雞蛋上之兩個小洞則用石蠟封堵。。 After all the eggs have been nicked and drilled, they are inoculated with virus using a tuberculin syringe – a 1 ml syringe fitted with a 1/2 inch, 27 gauge needle. The needle passes through the hole in the shell, through the chorioallantoic membrane, and the virus is placed in the allantoic cavity, which is filled with allantoic fluid. The two holes in the shell are sealed with melted paraffin, and the eggs are placed at 37 degrees C for 48 hours. During the incubation period, the virus replicates in the cells that make up the chorioallantoic membrane. As new virus particles are produced by budding, they are released into the allantoic fluid. To harvest the virus, the top of the egg shell – the part covering the air sac – is removed. We used to have a special tool to do this, which was placed over the egg. When the handle of this tool is squeezed, it makes a neat crack around the top of the egg. It was then easy to remove the flap of shell with tweezers. The shell membrane and chorioallantoic membrane are pierced with a pipette which is then used to remove the allantoic fluid – about 10 ml per egg. Sufficient virus may be produced in one or two eggs (depending on the viral strain) to produce one 15 microgram dose of vaccine.病毒顆粒釋放入尿囊液中。用小吸管每顆蛋吸取10cc尿囊液，大約每兩顆蛋可製出一劑15microgram的疫苗。 . Influenza hemagglutination inhibition assay 流感的血凝抑制分析 Centers for Disease Control and Prevention have determined that some adults have serum cross-reactive antibodies to the new influenza H1N1 virus. One of the techniques used to reach this conclusion is the hemagglutination inhibition (HI) assay. How does this assay work?將兩兩稀釋之含有病毒之血清和定量之紅血球放入小井中，若有病毒之存在，紅血球會形成Lattice格子狀結構而在小井表面形成有層膜，若無病毒存在，則紅血球會沈澱在小井底部。這種方法30分內會有結果、根據稀釋倍數，可半定量病毒數。這是叫血凝試驗。 To understand the HI assay, we must discuss the hemagglutination assay. Influenza virus particles have an envelope protein called the hemagglutinin, or HA, which binds to sialic acid receptors on cells. The virus will also bind to erythrocytes (red blood cells), causing the formation of a lattice. This property is called hemagglutination, and is the basis of a rapid assay to determine levels of influenza virus present in a sample. To conduct the assay, two-fold serial dilutions of a virus are prepared, mixed with a specific amount of red blood cells, and added to the wells of a plastic tray. The red blood cells that are not bound by influenza virus sink to the bottom of a well and form a button. The red blood cells that are attached to virus particles form a lattice that coats the well. The assay can be performed within 30 minutes, and is therefore a quick indicator of the relative quantities of virus particles. In the figure above, two-fold dilutions of samples of different influenza viruses (A – H) were prepared, mixed with chicken red blood cells, and added to the wells of a 96-well plate. After 30 minutes the wells were photographed. Sample A causes hemagglutination up to the 1:256 dilution; therefore the HA titer of this virus stock is 256. The sample in row B contains no detectable virus, while that in row D has an HA titer of 512. The HA assay can be easily modified to determine the level of antibodies to influenza virus present in serum samples. In the CDC study cited below, the authors wished to determine whether stored serum samples contained antibodies to the new influenza H1N1 strain. First they obtained a preparation of one of the new influenza viruses, specifically A/California/04/2009 and determined its HA titer by the method described above. They added a fixed amount of virus to every well of a 96-well plate, equivalent to 32 – 64 HA units. Then they prepared two-fold dilutions of each serum to be tested, and added each dilution series along a row of wells. Finally, they added red blood cells and incubated for 30 minutes.但若將血凝試驗加以modified,則可用來半定量病人血清中的抗體量！方法叫血凝抑制試驗！小井中放的是某特定病毒，約相當32至64HA單位，其餘都和血凝試驗同，病人血清中若有特定抗體存在，則會先和病毒結合，那紅血球就會沈澱至底部！CDC説只要血凝試驗，稀釋到1：40仍能和病毒作用，（即紅血球沈澱），就可降低感染率一半！讓血凝抑制試驗成為流行病學上一有力工具！ The basis of the HI assay is that antibodies to influenza virus will prevent attachment of the virus to red blood cells. Therefore hemagglutination is inhibited when antibodies are present. The highest dilution of serum that prevents hemagglutination is called the HI titer of the serum. If the serum contains no antibodies that react with the new H1N1 strain, then hemagglutination will be observed in all wells. Likewise, if antibodies to the virus are present, hemagglutination will not be observed until the antibodies are sufficiently diluted. The CDC report contains the statement “…serum HI antibody titers of 40 are associated with at least a 50% reduction in risk for influenza infection or disease in populations”. A serum HI antibody titer of 40 means that at a dilution of 1:40, the serum blocked hemagglutination. By determining HI titers and comparing them with influenza attack rates in populations, it is possible to calculate the significance of the HI antibody titer with respect to susceptibility to influenza virus infection. When used in this manner, the HI assay is a powerful epidemiological tool. J Katz, PhD, K Hancock, PhD, V Veguilla, MPH, W Zhong, PhD, XH Lu, MD, H Sun, MD, E Butler, MPH, L Dong, MD, PhD, F Liu, MD, PhD, ZN Li, MD, PhD, J DeVos, MPH, P Gargiullo, PhD, N Cox, PhD (2009). Serum Cross-Reactive Antibody Response to a Novel Influenza A (H1N1) Virus After Vaccination with Seasonal Influenza Vaccine Morbid. Mortal. Weekly Rep., 58 (19), 521-524 Influenza microneutralization assay 28 MAY 2009另一個CDC用來測定成人血清是否存在和新H1N1病毒有交叉反應抗體的試驗：流感微中和試驗。病毒感染常造成細胞病變效應：細胞變圓，從培養皿上脫落，利用血清中是否存在抗體、中和掉病毒，而不造成細胞病變效應。從血清稀釋的量來半定量存在的抗體。 The microneutralization assay is another technique used by the Centers for Disease Control and Prevention to determine that some adults have serum cross-reactive antibodies to the new influenza H1N1 virus. Let’s explore how this assay works. Viral replication is often studied in the laboratory by infecting cells that are grown in plastic dishes or flasks, commonly called cell cultures. Many viruses kill such cells. Here is an example of HeLa cells being killed by poliovirus: The upper left panel shows uninfected cells, and the other panels show the cells at the indicated times after infection. As the virus replicates, infected cells round up and detach from the cell culture plate. These visible changes are called cytopathic effects. There is another way to visualize viral cell killing without using a microscope: by staining the cells with a dye. In the example shown below, cells have been plated in the small wells of a 96 well plate. One well was infected with virus, the other was not. After a period of incubation, the cells were stained with the dye crystal violet, which stains only living cells. It is obvious which cells were infected with virus and which were not. We can use this visual assay to determine whether a serum sample contains antibodies that block virus infection. A serum sample is mixed with virus before infecting the cells. If the serum contains antibodies that block viral infection, then the cells will survive, as determined by staining with crystal violet. If no antiviral antibodies are present in the serum, the cells will die. In its present form, this assay tells us only whether or not there are antiviral antibodies in a serum sample. To make the assay quantitative, two-fold dilutions of the serum are prepared, and each is mixed with virus and used to infect cells. At the lower dilutions, antibodies will block infection, but at higher dilutions, there will be too few antibodies to have an effect. The simple process of dilution provides a way to compare the virus-neutralizing abilities of different sera. The neutralization titer is expressed as the reciprocal of the highest dilution at which virus infection is blocked. In the example shown here, the serum blocks virus infection at the 1:2 and 1:4 dilutions, but less at 1:8 and not at all at 1:16. Each serum dilution was tested in triplicate, which allows for more accuracy. In this sample, the neutralization titer would be 4, the reciprocal of the last dilution at which infection was completely blocked. This explanation should clarify how the neutralization titers were obtained that are reported in the CDC study cited below. By the way, microneutralization simply means that the neutralization assay is done in a small format, such as a 96 well plate, instead of larger cell culture dishes. The authors of the CDC study note that “although serum hemagglutination inhibition (HI) antibody titers of 40 are associated with at least a 50% reduction in risk for influenza infection or disease in populations, no such correlate of protection exists for microneutralization antibody titers”. They used mathematical analysis to determine the relationship between HI and microneutralization titers. They found that in sera from children, an HI titer of 40 corresponded to a microneutralization titer of 40. However, in adults, an HI titer of 40 corresponded to a microneutralization titer of 160 or more. I don’t know the reason for this difference, but one possibility is that not all neutralizing antibodies in adult sera are able to inhibit hemagglutination. Understanding why this situation might occur will require a discussion of how antibodies block viral infection. J Katz, PhD, K Hancock, PhD, V Veguilla, MPH, W Zhong, PhD, XH Lu, MD, H Sun, MD, E Butler, MPH, L Dong, MD, PhD, F Liu, MD, PhD, ZN Li, MD, PhD, J DeVos, MPH, P Gargiullo, PhD, N Cox, PhD (2009). Serum Cross-Reactive Antibody Response to a Novel Influenza A (H1N1) Virus After Vaccination with Seasonal Influenza Vaccine Morbid. Mortal. Weekly Rep., 58 (19), 521-524 Detecting viruses: the plaque assay 6 JULY 2009用plaque斑塊分析來測定病毒量。 One of the most important procedures in virology is measuring the virus titer – the concentration of viruses in a sample. A widely used approach for determining the quantity of infectious virus is the plaque assay. This technique was first developed to calculate the titers of bacteriophage stocks. Renato Dulbecco modified this procedure in 1952 for use in animal virology, and it has since been used for reliable determination of the titers of many different viruses.斑塊測定最早是用於噬菌體測試，1952年Renato Dulbecco將它修改用於動物病毒量之測試，沿用至今。 To perform a plaque assay, 10-fold dilutions of a virus stock are prepared, and 0.1 ml aliquots are inoculated onto susceptible cell monolayers. After an incubation period, to allow virus to attach to cells, the monolayers are covered with a nutrient medium containing a substance, usually agar, that causes the formation of a gel. When the plates are incubated, the original infected cells release viral progeny. The spread of the new viruses is restricted to neighboring cells by the gel. Consequently, each infectious particle produces a circular zone of infected cells called a plaque. Eventually the plaque becomes large enough to be visible to the naked eye. Dyes that stain living cells are often used to enhance the contrast between the living cells and the plaques. Only viruses that cause visible damage of cells can be assayed in this way. An example of plaques formed by poliovirus on a monolayer of HeLa cells is shown at left. In this image, the cells have been stained with crystal violet, and the plaques are readily visible where the cells have been destroyed by viral infection. The titer of a virus stock can be calculated in plaque-forming units (PFU) per milliliter. To determine the virus titer, the plaques are counted. To minimize error, only plates containing between 10 and 100 plaques are counted, depending on the size of the cell culture plate that is used. Statistical principles dictate that when 100 plaques are counted, the sample titer will vary by plus or minus 10%. Each dilution is plated in duplicate to enhance accuracy. In the example shown below, there are 17 plaques on the plate made from the 10-6 dilution. The titer of the virus stock is therefore 1.7 x 108 PFU/ml. Next we’ll consider how the plaque assay can be used to prepare clonal virus stocks, a step that is essential for studying viral genetics. Dulbecco, R., & Vogt, M. (1953). Some problems of animal virology as studied by the plaque technique. Cold Spring Harbor Symp. Quant. Biol., 18, 273-279 How many viruses are needed to form a plaque? 8 JULY 2009動物病毒，一個病毒顆粒就足以造成一個斑塊。 The plaque assay is an essential tool for determining virus titers. The concept is simple: virus infection is restricted to neighboring cells by a semisolid overlay. By counting the number of plaques, the virus titer can be calculated in PFU per ml. A key question is: how many viruses are needed to form a single plaque? For most animal viruses, one infectious particle is sufficient to initiate infection. This conclusion can be reached by studying the relationship between the number of infectious virus particles and the plaque count. A linear relationship means that one infectious particle can form a plaque. In this case the virus is said to infect cells with one-hit kinetics. This concept is illustrated below. In this figure, the number of plaques produced by a virus with one-hit kinetics or two-hit kinetics is plotted versus the relative concentration of the virus. There are some examples of viruses with two-hit kinetics: in other words, two different types of viral particles must infect a cell to initiate the infectious cycle. Examples include the genomes of some (+) strand RNA viruses of plants, which consists of two RNA molecules that are packaged in different particles. The dose-response curve of such viruses is parabolic rather than linear. When a single virus particle can form a plaque, the viral progeny within the plaque are clones. Virus stocks prepared from a single plaque are called plaque purified virus stocks. To prepare such virus stocks, the tip of a small pipette is inserted into the agar overlay above the plaque. The plug of agar is removed and placed in buffer. The viruses within the agar plug move into the buffer, which can then be used to infect cultured cells. To ensure purity, this process is usually repeated at least one more time. Plaque purification is used extensively in virology to establish clonal virus stocks. The ability to prepare clonal virus stocks was an essential development that permitted genetic analysis of viruses. Measurement of viruses by end-point dilution assay 13 JULY 2009 The plaque assay is a terrific method for determining virus titers, but it doesn’t work for all viruses. Fortunately there are several alternative methods available, including the end-point dilution assay. The end-point dilution assay was used to measure virus titer before the development of the plaque assay, and is still used for viruses that do not form plaques. Serial dilutions of a virus stock are prepared and inoculated onto replicate cell cultures, often in multi-well formats (e.g. 96 well plastic plates). The number of cell cultures that are infected is then determined for each virus dilution, usually by looking for cytopathic effect. In this example of an end-point dilution assay, 10 monolayer cell cultures were infected with each virus dilution. After an incubation period, plates that displayed cytopathic effects were scored with a +. At high dilutions, none of the cell cultures are infected because no particles are present. At low dilutions, every cell culture is infected. Half of the cell cultures showed cytopathic effects at the 10-5 dilution. This is the end point: the dilution of virus at which 50% of the cell cultures are infected. This number can be calculated from the data and expressed as 50% infectious dose (ID50) per milliliter. The virus stock in this example contains 105 ID50 per ml. In real life, the 50% end point does not usually fall exactly on a dilution as shown in the example. Therefore statistical procedures are used to calculate the end point of the titration. End-point dilution methods can also be used to determine the virulence of a virus in animals. The same approach is used: serial dilutions of viruses are made and inoculated into multiple test animals. Infection of the animal can be determined by death or clinical symptoms such as fever, weight loss, or paralysis. The results are expressed as 50% lethal dose (LD50) per ml or 50% paralytic dose (PD50) per ml when lethality or paralysis are used as end points. The following example illustrates the use of end point dilution to measure the lethality of poliovirus in mice. Eight mice were inoculated per virus dilution, and the end point was death. The statistical method of Reed and Muench was used to determine the 50% end point. In this method, the results are pooled, and the mortality at each dilution is calculated. The 50% end point, which falls between the fifth and sixth dilutions, is calculated to be 10-6.5. Therefore the virus sample contains 106.5 LD50 units. Reed, L.J., & Muench, H. (1938). A simple method of estimating fifty percent endpoints. Am. J. Hygiene, 27, 493-497

再談流感

流感是一種黏液病毒屬於正黏液病毒科（family),本只局限在水禽、但在六至九千年前傳播至人類，正好和農耕、畜牧及都市化的時間點吻合。 人類行為的改變、人口的集中提供病毒、像流感、麻疹、天花、從動物變成可適應、感染人類。 流感數世紀以來廣為認知，但真正證實是病毒致病則是在1930年代的英國。流感英文為influenza,是源自義大利文，意思是＂影響＂、在中古世紀認為是因星球不正常的排列、造成惡劣氣候的影響。現在看來這種想法當然荒謬。但即使在中古世紀、人們也認知到有些疾病會一個傳一個，若是星相的影響，則無法解釋為何不是同時一起發病，而是一波一波。 在1892年流感大流行時，微生物學家Pfeiffer從死於流感病患肺中分離出一種新的細菌，他稱之為Hemophilus influenza.隨後大家都能從流感合併肺炎患者分離出此菌，大家一至認為此菌就是流感的病原。直到1918年又發生全球大流行，醫學界苦思想要製造疫苗來預防，但雖許多病患身上可分離出嗜血流感菌，但也有相當多患者分離不出，當時認為是培養技術不佳，雖然Peter Olitsky and Frederick Gates at The Rockefeller Institute.將病人鼻液用一種細菌無法通過的過濾器濾過，而過濾液仍能使兔子肺炎。但其他人重複實驗卻無法得出相同結果，再加上Oswald Avery (Rockefeller Institute）發展出chocolate agar 使得嗜血流感菌培養陽性率大增，讓眾人更深信不疑流感是由嗜血桿菌所引起的。 直到同在Rockefeller institute 研究豬隻霍亂的Shope,注意到1918、，1929在豬隻養殖廠發生兩次豬瘟大流行，症狀與人流感相似，於是嘗試從病豬檢體培養，果然也是長出嗜血桿菌，但是將此菌注射至一般豬隻、並無法出現豬瘟！但是檢體的過濾液卻能致病！表示致病因素在過濾液中。 1930年代，英國的國家醫學研究機構的三人，Wilson Smith, Christopher Andrewes, and Patrick Laidlaw (National Institute for Medical Research, UK）則從過濾液中分離出病毒，且證實其致病性。而且發覺1918年流感痊癒病人的血清具有可以中和此豬流感病毒作用，後證實1918那株病毒其實一直在豬隻中傳播至今！ 1932電子顯微鏡的上市，1930年代分子生物學的進步，了解到流感的致病原是病毒！ 流感病毒的蘆山真面目 屬單負股的RNA病毒，正黏液病毒科，外包覆著源自宿主，富含脂質的envelope,內有由Capsid保護著的,8節段病毒RNA..（PA,PB1,PB2,HA,NP,NA,M,NS),總基因長約14000個核甘，載有11種基因，是14種蛋白質的基因密碼所在。人基因有30億個核苷，大理石肺魚(marbel lungfish)有1500億個核苷。因是負股RNA,就必需先複製成正股RNA才能進行轉譯為蛋白質。 而把負股RNA複製成正股所需之聚合酶，病毒需自備，共有3種：PB1,PB2,PA. 聚合酶複製能力的強弱決定宿主病情之輕重。 流感病毒是使用血凝素和宿主細胞表面含唾液酸的受器結合而進入細胞內部，這是最關鍵的步驟，若有抗體對付血凝素、則宿主不會被感染。病毒表面還有另一蛋白叫神經氨酶，它可以將宿主呼吸道上皮表面的黏液加以分解，方便受器的露出，以利血凝素的附著、結合。另外在病毒裝配完成，即將脫離宿主時也需神經氨酶解開。克流感就是抑制神經氨酶而達到抑制病毒複製的目的。 PB2:是負責把宿主mRNA的cap加以分解、及合成引子primer PB1:是病毒聚合酶的核心亞單位 PB1-F2:會結合宿主細胞的粒線體、造成細胞凋亡，引發病變。 PA:是病毒聚合酶的亞單位，具蛋白酶功能，但真正作用不明。 HA:用來和宿主受器（唾液酸）結合 NP:不怕RNA酶之分解之蛋白質，和病毒RNA結合後，幫助其由細胞核移動至胞質中，也參與病毒之包裝及裝配。 NA:切斷唾液酸sialic acid,協助病毒之脫離 M1:在envelope下方形成一coating包住病毒RNA M2:為跨膜離子通道，使質子得以進入capsid,使胞內酸鹼質變酸，導至M1的不穩定，而使病毒RNA得以分離 NS1:抑制宿主mRNA出核，相對使病毒mRNA易出核，使宿主基因不表達干擾素，抑制T細胞的活化 NS2/NEP:協助病毒mRNA出胞核 流感病毒的命名格式如下: 型別/來源區域/病毒株號/分離年份(HN)。如：A/Taiwan/1/1986(H1N1)，表示它是1986年在台灣分離出而具有H1N1亞型的A型流感病毒，其病毒株編號為1。 B/Hong Kong/330/2001 ，表示它是2001年在香港分離出的B型流感病毒，其病毒株編號為330。 流感病毒是一種成功的病毒，因其不斷的變異，以及當不同株病毒同時感染同一素主時、可發生重組現象。RNA聚合酶不具備校誤之功能，所以約每1萬個核苷會出現一個錯誤，而流感病毒的基因組約1.4萬，所以每次複製約出現1至2個錯誤。複製越多，累積錯誤越多，人和黑猩猩的基因只相差1%，但同一宿主所分離出之病毒的基因可以相差4%.此種基因變異稱antigenetic drift抗原漂移，這種情況多發生在每年，造成地區性，季節性的流行。但若是整條RNA和其它株病毒發生交換，稱為 antigenetic shift抗原遷移，這種情況大約每20-30年發生一次，但會造成全球性大流行。 流感有A,B,C三型，C型原則對人不致病，B型只感染人，但A型流感可感染哺乳動物，鳥類。A型流感幾乎都來自禽類，它在野生水禽的消化系統中生存，但並不致病。因而可不斷隨著其遷徙時不斷傳播。也因其可同時感染哺乳動物，禽類，所以20世紀幾次大流行都是源自東亞、東南亞，因為此地區生活習慣常是人、獸、禽雜處。 對病毒抗原尤其是血球凝集素所產生的免疫反應，可以減少被感染的可能性，即使被感染也可減輕疾病嚴重度。但是對某一病毒株或其亞型產生的抗體，對其他病毒株或其亞型並無法提供或僅能提供部分保護作用。同樣的，如果病毒出現新的變異，原有的抗體也無法完全發揮保護作用。 歷史上的大流行 人類近代史上有三次流感大流行，皆由A型流感病毒引發，每一次都造成龐大的死傷。(1)1918年西班牙流感：1918年至1919年，H1N1型流感分別在法國、美國波士頓及非洲開始爆發，在全球傳播，導致罕見的嚴重性及致死率，達百分之2，死亡人數約2000至4,000萬。死亡曲線呈W型，而非往年的U型，中間多一高峰，死亡者有許多是二十至三十歲，身體健壯的年輕人，這和往常死者多5歲以下，65歲以上情況不同！但十八個月後疾病突然消失。 (2)1957年亞洲流感：1957年至1958年的H2N2型流感，死亡人數估計為200萬。 (3)1968年香港流感：1968年至1969年的H3N2型流感，死亡人數估計為100萬。 隨著科技的進步，目前已有能力還原出1918年西班牙流感的病毒基因序列，科學家從三位受害者保留下來的肺部組織中，將史上死傷最慘重的1918年流感病毒在實驗室內重生(resurrection)，並成功的感染實驗動物及造成100%的致死率，於是拼湊出完整面貌。重現這病毒，讓科學家研究它高致死率原因，追查其起源。 新型流感 流感病毒之基因容易突變，並可存在於多重宿主，當不同來源的流感病毒發生重組，常使抗原發生重大改變，或因不明原因，造成症狀及感染宿主發生變化之新病毒，即為「新型流行性感冒(novel influenza)」之概念。 在防治上，「新型流感」係指民眾就醫後，其臨床症狀、流行病學資料符合採檢條件，並經檢驗結果確認其為A型流感病毒，且亞型為H1、H3以外者。人類感染禽流感亦屬於新 型流感之一。 流感之實驗室診斷 (1)病毒培養： 傳統上，取鼻咽或喉頭拭子檢體於雞胚胎培養，缺點為費時，約需3至10天。 (2)檢測病毒RNA： 利用反轉錄聚合酉每鏈反應(RT-PCR)等分子生物學檢驗方法，可較快且準確地作出診斷。結果可在1天內得知，是目前最受歡迎的檢測方法。 (3)快速抗原診斷法: 取鼻咽或喉頭拭子檢體，檢測流感病毒抗原，可在30分鐘內得知結果，但其敏感性及專一性較差。 (4)檢測血中抗體： 必須檢測急性期與恢復期血清，比對是否抗體陽轉或4倍以上上升，需兩週以上才可得知結果，因此通常只用於流行病學之調查。 禽流感 禽流感是由禽型流行性感冒病毒感染所引起，所有的禽型流行性感冒病毒都是A型流感病毒，宿主有雞、鴨、鵝、水鳥、鯨魚、海豹及水貂等。病毒在自然宿主野鴨的消化道表皮細胞繁殖，但不致病，僅在糞便內排出病毒，且可達兩個星期之久。其他家禽若接觸到此種糞便，便可能吸入病毒至呼吸道內而發病。一般而言，禽流感病毒可分為高致病性和低致病性兩種，受高致病性禽流感病毒感染的鳥類，家禽致死率可高達80%以上。最近被高度注意的高致病性禽流感病毒主要為H5N1流感病毒，此禽流感病毒亞型最初於1961年在南非從鳥類(燕鷗)中首次分離。H5N1病毒在鳥類中傳染性非常強。 H5N1病毒在人類身上的增生效率不佳, 直接傳給人類相當罕見。然而近年來情況卻悄悄地發生了變化。禽流感的傳播已經跨越了原先的物種範圍，開始侵襲人類，而其高致死率也引起廣泛的注意。人類感染禽流感後，潛伏期一般為2-5天，症狀與其它流感相似，主要為發燒、流鼻涕、鼻塞、咳嗽、咽痛、頭痛、全身不適，有些會有噁心、腹痛、腹瀉、稀水便等消化道症狀，超過6成的患者可見肝指數 異常，病情嚴重者會發展成進行性肺炎、急性呼吸窘迫症、肺出血、腎衰竭、敗血症休克等多種併發症，甚至死亡。死亡率高達3分之2！ 禽流感病毒在禽類造成的大流行在文獻上並不少見，而造成人類的感染則是到1996年才首次被發表出來。隔年(1997年)，在香港即發生18例人類感染禽流感病毒(H5N1)，其中6人死亡。 2005年1月起，發生在越南的個案，其流行病學有了明顯的改變： 病例集中在北部，年齡層變大，病例持續發生，暗示著傳染方式的多樣性以及病毒在環境中存在的時間延長；而死亡率的降低，甚至出現無症狀的感染個案，則反映著病毒正逐漸調適中。而病毒學的分析也顯示，這些從人類分離出來的H5N1病毒都來自禽類，並未發生基因重組的現象。 但2005年分離出來的病毒已有抗原微變(antigenic drift)的情形，這代表病毒正逐漸調適人類細胞的接受器。 台灣目前並無發現高致病性的H5N1病毒，2003年於彰化縣及嘉義縣養雞場檢出之病毒均為H5N2家禽流行性感冒弱毒株。 20世界的幾次全球大流行： Major flu pandemic strains of the twentieth century： Year Designation Common name 1900 H3N8 (none) 1918 H1N1 Spanish flu西班牙 1957 H2N2 Asian flu亞洲 1968 H3N2 Hong Kong flu香港 1977 H1N1 Russian flu蘇聯 1997 H5N1 Avian flu禽

Croup

哮吼 (Croup) 哮吼是一種因為喉部阻塞而引起吸氣性喘鳴 (inspiratory stridor)， 聲音嘶啞， 吠狀咳嗽 (barking cough) 、胸骨上凹陷 (suprasternal retraction) 及呼吸困難等臨床表現的症候群，感染性哮吼的主要原因為會厭炎、病毒性哮吼與細菌性氣管炎。雖然哮吼這個診斷名詞主要是描述喉部的變化，但是其感染部位也常常延伸到呼吸道的其他地方。所以血氧值低下、囉音、肺部浸潤的X光顯像等臨床表現，在哮吼患者並不罕見。 會厭炎 (epiglottitis)：大多發生於 2-7 歲兒童，幾乎均由 b型嗜血桿菌 (Hemophilus influenzae type b) 引起， 台灣地區很少發生此種病例。它通常是急性發作，病人除了高燒以外，會呈現虛脫與毒性病容，常常因為嚴重的呼吸道阻塞而無法躺下，並出現頸部後仰與下巴前傾的姿勢。嚴重時可導致發紺， 休克及意識昏迷。一般比較少出現聲音沙啞與吠狀咳嗽，所以會厭炎並非典型的哮吼。理學檢查時， 將舌頭壓下可以看到明顯腫大呈桃紅色的會厭軟骨。這種壓舌檢查有人認為會引起反射性喉部痙攣和吸入分泌物而導致急性呼吸阻塞， 甚至死亡。所以對於懷疑為此症的病人，最好先作頸部側面的 X光檢查，如果有必要直接壓舌檢查， 應該先備好急救設備。因為此症常常引起厲害的呼吸道阻塞而危及生命， 所以在確定診斷之後，必須馬上住進加護病房，施行氣管插管或氣管切開術， 並給予抗生素 7-10天，可以選用第二， 三代頭芽胞菌素 (cephalosporin)、amoxicillin-clavulanic acid 或 ampicillin-sulbactam。 病毒性哮吼 (viral croup，subglotittis)：通常發生於三個月到五歲的小孩子，由病毒引起， 特別是副流感病毒，其他還包括流感病毒、呼吸細胞融合性病毒、腺病毒等。一般發病比會厭炎緩慢而且症狀較輕微， 大多先有一些上呼吸道感染的症狀，一至數天之後才有哮吼的典型表現， 頸部前側的X光檢查可以看到會厭下的氣道縮窄，而出現所謂的尖塔徵象 (steeple sign)、砂漏徵象 (hourglass sign) 或肩部徵象消失(loss of shoulder sign)。X光的檢查僅供參考，臨床的診斷主要還是依據病人症狀的表現。治療上可以給予氧氣、濕氣等一般支持性療法。症狀比較嚴重者，給予類固醇可以縮短病程，但是在使用之前必須先排除下述細菌性氣管炎的可能性，以免反而拖延了病程。 細菌性氣管炎 (bacterial tracheitis)：通常發生於三歲以下兒童，在台灣的哮吼病人之中，是僅次於病毒性哮吼的第二常見原因。常見的致病菌是金黃色葡萄球菌、viridans streptococcus、嗜血桿菌與 Moraxella catarrhalis。臨床表現和 X 光檢查與病毒性哮吼十分類似，發病也是先有一些上呼吸道感染的症狀， 一至數天之後才有哮吼的典型表現， 頸部前側的 X光檢查，也呈現會厭下的氣道縮窄。與病毒性哮吼不同的是，病人的發燒比較厲害，甚至有毒性病容，實驗室檢查常常出現白血球計數上升與不成熟白血球比例增加的現象，確定的鑑別診斷必須依靠支氣管鏡檢查。這種檢查除了可以確定會厭下方的氣管有化膿現象以外，也可以直接做培養以鑑定菌種。治療上必須給予抗生素，例如第二代頭芽胞菌素 (cephalosporin)、amoxicillin-clavulanic acid、ampicillin-sulbactam，可加aminoglycoside，病情嚴重者常常需要做氣管插管，並使用呼吸器。

美國最佳飲食法 ：降血壓飲食法 DASH Diet 健康飲食法 減肥飲食法 美國一項最新排名顯示，降血壓飲食法（DASH Diet）成為2012年美國最佳飲食法，這項評選是由U.S News & World Report做出的。 DASH是停止高血壓的膳食方法的英文首字母縮寫，該方法的創始人發明者宣稱這是最健康的飲食方法，也是最好的糖尿病飲食法。 不過，即使在美國本土，這個計劃也甚少人知之。難道是因為它有什麼副作用，或者像阿金飲食法那樣會引起輕度營養不良嗎？ 我們一起了解一下這個新的健康計劃吧。如果你想知道怎樣開始和堅持這個計劃，以及健康飲食為何無法保證減肥等等，請看下面的降血壓飲食法五個要點。 1. 基本原理 降血壓健康計劃最初是一種降低血壓的方法：鼓勵人們吃更多蔬菜和水果，減少肉和脂肪的攝入，並將抗血壓的元素綜合在內。 通過將卡路裏總量降為負值，該飲食計劃也成為一個有效的減肥方法並受到美國人的關注。 這個計劃要求你多吃水果、蔬菜以及低脂肪的食物、全麥食物、魚和家禽等，減少食用高脂肪和膽固醇的食物以及紅肉、甜食和鹽。 除了這些基本食物以外，降血壓飲食法也將豐富的食物加入計劃中，稱之為"關注營養"，比如美國人飲食中較為缺乏的鈣和纖維。 "關注營養"包括可消化的纖維、幫助降低血壓和減少骨質流失的鉀、強健骨骼、血管和肌肉的鈣、有助於細胞代謝的維生素B-12以及調節身體中的鈣和磷酸鹽的維生素D。 雖然降血壓飲食法沒有提供一個精確的計劃，但它提供各種水平和健身目標的建議，並鼓勵更加積極的生活方式。 2. 開始很容易...... 降血壓飲食法的書籍受到了節食者們的歡迎，其中每日卡路裏計算是一個簡單易行的計劃。 "降血壓飲食法是合理、健康、有效的，"一名讀者在該飲食法的官方網站上留言說，"書很容易懂，也容易操作。" 其他讀者也稱贊關於這種飲食法的書指出了不同卡路裏水平的分量，一些人說這是他們過去所困惑的。"這是第一個合理的飲食法。"一位讀者寫道。 想要了解這個飲食法的讀者不需要花錢買書，你可以在這裏下載免費的指導，了解應該吃什麼，以及根據你的年齡和運動水平確定的每日卡路裏數量。 3. 不過，難以堅持...... 不過，困難的是，這個飲食法在改變人們的飲食習慣，說起來要比去做容易得多。 降血壓飲食法要求改變人們的飲食規律，如果想每天堅持的話需要下很大的功夫。 比如，五月診所（May Clinic）的降血壓飲食法指南甚至對購物做了規定。除了每周開始制定詳細的購物清單之外，購物者還需仔細查看營養標簽上的卡路裏、脂肪、維他命、礦物質和蛋白質含量。這裏沒有提前做好的食物或者每周免費派送。 但除了繁重的工作以外，還有價格的問題。對於節食者或者那些只是想要更加健康的人來說，購買所有新鮮或者有機的食物是一大筆開支。即使在外面餐館就餐，昂貴的價格可以抵消掉讓別人為你做飯的喜悅。 一項美國調查顯示，各種飲食法由於要求繁重的工作及昂貴的價格而難以普及。 4. 降血壓飲食法容易按需定制 降血壓飲食法沒有固定的套路，但好處是可以變得多樣化。這個計劃強調食物中顏色、質感和香味的多樣化。與別的健康計劃不同，新的飲食法可以容易地被猶太教、伊斯蘭教或無麩質飲食以及素食或者嚴格素食的追隨者采納。 實際上，據其官網介紹，降血壓飲食法最初是模範素食主義者的飲食開發的，因為與雜食者相比，素食主義者一般食用更少的食鹽、血糖較低。 5. 這是一個健康飲食法，不是減肥飲食法 降血壓飲食法無疑是一個最健康的計劃。除了適度減肥和降低血壓之外，這個飲食法可能也有助於降低膽固醇。據內科醫學檔案2008年的一項研究，節食與降低中年婦女冠心病和中風的幾率相關。 22名志願者組成的專家組將降血壓飲食法評選為2012年最佳飲食法的原因之一是，它能幫助長期和短期減肥，效果顯著。這個飲食計劃目前沒有發現存在健康風險，並且看起來可以產生多種健康益處。 要記住的最重要一點是，這個計劃最初並非為減肥而設計，那些想要減掉很多磅的人需要著眼於這個計劃的長期效果。 由於降血壓減肥法拒絕低碳水化合物，把重點放在運動和改變人們的食物觀念上，其減肥的收效時間要比液體飲食法、阿金飲食法等花更長時間。多吃一點水果和蔬菜不會讓船沉下去，但吃很多要過和杏仁--由於這個飲食法鼓勵吃堅果--則會增加每天的卡路裏攝入量，盡管食物是健康的。

國際素食日的省思！

11月25日是國際素食日，雖然此節日來源於印度，但是由於其健康的生活方式得到了越來越多人的認同，終發展為一個世界性的節日。趁著節日，今天小編就給大家講講素食的問題，希望大家正確對待素食。 完全素食並非養生最佳辦法 為什麼“國際素食日”如此受歡迎呢？其實是與現代社會高發的“富貴病”有關。由於過多食用動物性食品，很多人都出現了高血壓、糖尿病等問題，因此，很多人對素食越來越感興趣了。甚至有人認為，一切疾病都是由於過多食用葷性食物造成的。因此有的人甚至成為了完全的素食主義者，這樣真的會健康嗎？ 素食，即植物性食品可以降低膽固醇和飽和脂肪酸的攝入量，防止膽固醇進入血液，減少肥胖病、高膽固醇血症和冠心病等的發生，保持人體酸堿平衡。另外，素食中含有豐富的維生素，可以調節代謝功能，加強皮膚的營養。如此說來，素食似乎是養生的最好途徑了。其實不然。雖然小編也主張大家平時要多吃素食，但是卻並不贊同完全素食的行為，在今天大家清清腸胃可以，但是完全素食卻並非養生的最好辦法。 國際素食日：素食養生亦存隱患 最新研究表明，素食能養生，卻不利於強身健體，如果長期吃素食，非但不會對身體有益，還有可能導致孩子弱智、女性閉經等各種疾病的後果。由於食物單調，素食者機體中掌管食物消化的酶系統的功能逐漸遭到破壞，最後導致物質交換失調，疾病叢生。 雖然植物性食品中富含維生素、無機鹽類和有機酸，但缺少造血的微量元素鈷、錳、鐵和銅等。此外，植物性食品除油料外，脂肪含量極少，但人的機體每晝夜至少需要60-70克脂肪，而且，植物蛋白永遠代替不了動物蛋白。因此，專家建議只有在特殊情況下，素食才可作為臨時性飲食措施。 長期素食，蛋白質得不到充分供給，其後果是記憶力下降，精神委靡，反應遲鈍。臨床醫學發現，蛋白質不足是引起消化道腫瘤和胃癌的一個重要原因。另外，人腦的形成發育所必需的大部分營養成分必須從動物性食物中攝取，如缺乏則可導致人腦退化，患癡呆症。 由於單純素食無法得到維生素B12，而機體缺乏維生素B12，可導致精神和心理上的缺陷，如表情呆滯，舌頭腫痛，吞咽困難，容易疲勞等。孕婦若長期素食可導致胎兒巨幼紅細胞貧血和腦組織永久性損傷。因此，注意葷素搭配才是最健康的養生法。 專家：2分葷6分素最健康 現代營養學家認為，植物性食品和動物性食品比例合理地結合起來，才能滿足機體生長發育和生活的需要。因此，人們應當放棄素食養生的傳統觀念，單純素食不可取，只有葷素搭配營養全面，平衡膳食，才能有一個健康的身體。那麼如何吃飲食最健康呢？ 錯誤的飲食習慣，人們傾向於食用更多的動物性食物，結果導致了葷素比例的失衡，成了7分葷1分素，這樣不但破壞了健康的飲食結構，還會影響到營養的吸收。專家建議從營養學角度來說，人類飲食的葷素黃金比例應該為2：6，即2分葷6分素。