Reid
Ann H. Reid. (Volver a Personas. Cosas. Geografía. Cronología. Fuentes.)
Coautora, militar, con tres conocidos autores, en 1999 habla de 20 millones de muertos, cuatro años más tarde, en cooperación con otro de esos autores, habla ya de 40 millones.
Proc Natl Acad Sci U S A. 1999 Feb 16; 96(4): 1651–1656. PMCID: PMC15547 Microbiology Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene Ann H. Reid,* Thomas G. Fanning, Johan V. Hultin, and Jeffery K. Taubenberger.
Division of Molecular Pathology, Department of Cellular Pathology, Armed Forces Institute of Pathology, Washington, DC 20306-6000
- To whom reprint requests should be addressed. e-mail: reid@afip.osd.mil
Communicated by Edwin D. Kilbourne, New York Medical College, Valhalla, NY
ABSTRACT The “Spanish” influenza pandemic killed over 20 million people in 1918 and 1919, making it the worst infectious pandemic in history. Here, we report the complete sequence of the hemagglutinin (HA) gene of the 1918 virus. Influenza RNA for the analysis was isolated from a formalin-fixed, paraffin-embedded lung tissue sample prepared during the autopsy of a victim of the influenza pandemic in 1918. Influenza RNA was also isolated from lung tissue samples from two additional victims of the lethal 1918 influenza: one formalin-fixed, paraffin-embedded sample and one frozen sample obtained by in situ biopsy of the lung of a victim buried in permafrost since 1918. The complete coding sequence of the A/South Carolina/1/18 HA gene was obtained. The HA1 domain sequence was confirmed by using the two additional isolates (A/New York/1/18 and A/Brevig Mission/1/18). The sequences show little variation. Phylogenetic analyses suggest that the 1918 virus HA gene, although more closely related to avian strains than any other mammalian sequence, is mammalian and may have been adapting in humans before 1918.
The influenza pandemic of 1918 was exceptionally severe, killing 20–40 million people worldwide, with unusually high death rates among young, healthy adults (1). A mild wave of influenza in the spring and summer of 1918 was highly contagious but caused few deaths. In late August, a virulent form of the disease emerged and swept the globe in 6 months, killing over 10,000 people per week in some U.S. cities (1) at the height of the pandemic.
Recently, this laboratory reported the isolation of fragments of RNA from the 1918 influenza virus from preserved lung tissue of a victim of the deadly fall wave of the pandemic. Sequence from 5 of the virus’s 10 genes indicated that the strain was of the H1N1 subtype and different from any other sequenced influenza strain. All of the sequenced genes were more similar to those influenza strains infecting mammals than to those that infect birds (2). We now report the complete sequence of the hemagglutinin (HA) gene of the 1918 influenza virus. The full-length sequence was generated by using RNA fragments isolated from the victim reported in March 1997 [A/South Carolina/1/18 (H1N1)] (2). Additional sequence was obtained from two recently identified 1918 victims.
Influenza is a zoonotic disease, affecting many species of birds and mammals. The HA protein is found on the surface of the influenza virus particle and is responsible for binding to receptors on host cells and initiating infection. HA is also the principal target of the host’s immune system. Thus, in order for influenza to spread in a new host, the HA protein must acquire the ability to bind to the new host’s cells. Once in the new host, the HA protein comes under selective pressure for change to evade the host’s immune system (3).
Pandemic influenza results when an influenza strain emerges with an HA protein to which few people have prior immunity (4). It is thought that the source of HA genes that are new to humans is the extensive pool of influenza viruses infecting wild birds. Of the 15 HA subtypes found in birds, only 3 (H1, H2, and H3) are known to have caused pandemics in humans (5). Recently an H5 virus caused illness in a limited number of people in Hong Kong, China (6, 7). How new HA genes emerge and where they adapt from their characteristic avian form to a form that successfully spreads in humans are not understood perfectly. In both 1957 and 1968, the pandemic strains had new HA genes that were very similar to known avian strains (8, 9). We sought to determine whether the HA gene of the 1918 pandemic strain also had avian characteristics.
Go to: MATERIALS AND METHODS Case Selection. Autopsy cases of 78 victims of the lethal fall wave of the 1918 pandemic were examined for this study. Evidence from 74 victims consisted of formalin-fixed, paraffin-embedded tissues, stained slides, and clinical records from the files of the Armed Forces Institute of Pathology. All the samples were screened by histologic analysis. The majority of individuals died of secondary acute bacterial pneumonia, the most common cause of death in the 1918 pandemic (10); most of the samples taken from these individuals were not analyzed further, because they were extremely unlikely to retain influenza virus (11, 12). However, 13 samples were selected by histologic and clinical criteria for further analysis. These samples were from patients who experienced acute influenza deaths after clinical courses of less than 1 week. In addition to samples taken from patients with early bronchopneumonia, samples from patients with acute massive pulmonary edema and/or hemorrhage were also selected, reflecting the unusual histopathology observed in 1918 (13). Of these 13 samples, 2 were positive for influenza RNA on subsequent molecular genetic analysis.
Case Histories. The first patient was a 21-year-old male stationed at Ft. Jackson, SC. He was admitted to the camp hospital on September 20, 1918 with influenza and pneumonia. He had a progressive course with cyanosis and died on September 26, 1918. During the autopsy, it was noted that he had a fatal secondary lobar bacterial pneumonia in his left lung, whereas the right lung showed only focal acute bronchiolitis and alveolitis, indicative of primary influenza pneumonia. Formalin-fixed, paraffin-embedded right lung tissue was positive for influenza RNA [A/South Carolina/1/18 (H1N1)] as reported (2).
The second patient was a 30-year-old male stationed at Camp Upton, NY. He was admitted to the camp hospital with influenza on September 23, 1918, had a very rapid clinical course, and died from acute respiratory failure on September 26, 1918. The autopsy showed massive bilateral pulmonary edema and focal acute bronchopneumonia. Formalin-fixed, paraffin-embedded lung tissue was positive for influenza RNA [A/New York/1/18 (H1N1)]. RNA templates larger than 150 nucleotides could not be amplified in these two cases.
An additional 1918 influenza case was found by examining lung tissue from four 1918 influenza victims exhumed from a mass grave in Brevig Mission on the Seward Peninsula of Alaska. Brevig Mission (called Teller Mission in 1918) suffered extremely high mortality during the influenza pandemic in November 1918. Although individual case records were not available, historical records show that influenza spread through the village in about 5 days, killing 72 people, representing about 85% of the adult population (1, 14). Victims were buried in a mass grave in permafrost. In August 1997, four of these victims were exhumed. Frozen lung tissues were biopsied in situ from each, and tissues were placed in formalin, alcohol fixatives, and RNAzol (Tel-Test, Friendswood, TX). Although the histologic analysis was hampered by artifacts of freezing, these tissues showed evidence of acute massive pulmonary hemorrhage and edema. One of the victims, an Inuit female (age unknown) was influenza RNA positive [A/Brevig Mission/1/18 (H1N1)]. In this case, RNA templates greater than 120 nucleotides could not be amplified.
Avian HA1 proteins have changed less over time than human HA1 proteins. Swine HA1 proteins have an intermediate number of changes. For example, the 1918 HA1 has just 4 changes from the putative mammalian ancestral sequence, whereas the closest swine sequence differs by 10 changes from the same ancestral sequence. The 1918 HA1 has 13 changes from the putative avian ancestor, whereas the closest avian sequence differs by 8 changes from that ancestor.
Finally, we performed parsimony analysis by using only the three 1918 cases and nine avian strains to see whether the 1918 cases are placed in the mammalian clade, because all mammalian H1 strains are derived from a strain similar to the pandemic virus. This analysis showed the presence of two clades, avian and 1918, suggesting that the 1918 sequences are indeed phylogenetically distinct from avian strains.
Plotting the total number of amino acid changes from node to node (Fig. (Fig.3)3) vs. the year of isolation for viruses in the human clade before 1954 suggests that the ancestor of the 1918 virus entered humans around 1900. Tracing total numbers of nucleotide changes from node to node of a nucleotide parsimony tree places the 1918 ancestor in humans around 1915.
The 1918 strains, in contrast, have only the four glycosylation sites shared with avian and swine strains (Fig. (Fig.1), 1), suggesting that acquisition of additional glycosylation sites is not required for efficient replication in humans.
Antigenic Sites. On the H1 protein, four antigenic sites, encompassing 42 amino acids, have been identified (refs. 25 and 28; Fig. Fig.1).1). In human strains, these are among the most variable amino acids in the HA protein. At the Sa site, the 1918 strains match the avian sequences at all 12 amino acids. At the Sb site (9 amino acids), the 1918 strains share the avian consensus at 8 amino acids, but at amino acid 156, the 1918 strains have a serine that is found only in one other H1 strain (A/Sw/Illinois/63). At the Ca site (7 amino acids), the 1918 sequences have one change, N74S, from the avian consensus. This change is found in all swine strains but not in any human strains. At the Cb site (13 amino acids), the 1918 sequences differ from the avian consensus by two amino acids, S139A and T166V. The first change is found in all swine and some early human strains, and the second is found in all swine and human strains. Overall, the antigenic sites resemble the avian consensus closely, suggesting that there may have been little selective antigenic pressure on the HA protein before 1918.
DISCUSSION Previous phylogenetic analyses of H1 sequences suggest that the swine and human lineages diverged from a common, avian-like ancestor around 1905 (29, 30). Other analyses, which propose a higher rate of mutation early in the adaptation of avian influenza strains to mammalian hosts, suggest that the pandemic virus derived from an avian virus immediately before 1918, possibly through a swine intermediary (31, 32).
Of all mammalian H1s, those of the 1918 viruses are most similar to their avian counterparts. However, the 1918 HA gene has accumulated enough nonselected, mammal-associated changes to place it consistently in the mammalian clade phylogenetically (Fig. (Fig.2).2). The 1918 HA1 sequence differs from its closest avian relative by 26 amino acids, whereas the 1957 H2, the 1968 H3, and 1997 H5 HAs had 16, 10, and 3 differences, respectively, with their closest avian relatives (6–9). Phylogenetic analyses of the 1957, 1968, and 1997 HAs place them in avian clades and suggest that all three strains derived from the Eurasian group of avian strains. By contrast, phylogenetic analyses invariably place the 1918 sequence outside the avian clade and suggest that the 1918 sequence is phylogenetically equidistant from the Eurasian and North American avian strains.
Although the 1918 HA always falls within the mammalian group, it will sometimes seem to be more human-like, sometimes more swine-like, depending on the method of analysis. These results may be explained, at least in part, by the widely differing substitution rates in different lineages making up the tree (see Fig. Fig.3).3). Human strains, because of strong immune selection, have many amino acid substitutions that are not shared with the 1918 strains or swine strains. As a result, phylogenetic analyses based on amino acid sequence or nonsynonymous nucleotide substitutions may tend to group 1918 and swine strains together. Synonymous substitutions, on the other hand, reflect drift unrelated to immune selection and indicate a closer relationship between 1918 strains and subsequent human strains. In all of the phylogenetic analyses, branch lengths near the root of the tree were very short, suggesting that there may be few differences between the 1918 sequences and the common mammalian influenza virus ancestor.
The existing strain to which the 1918 sequences are most closely related is A/Sw/Iowa/30, the oldest classical swine flu strain. It has been known since the 1930s that survivors of the 1918 influenza had antibodies that neutralized classic swine influenza virus (33), and A/Sw/Iowa/30 is very similar to the 1918 strains at the antigenic sites. Because individual swine do not live long enough to exert immune selective pressure on the virus, influenza drifts more slowly in swine than in humans (30). Therefore, one would expect a swine virus isolated in the 1930s to retain more characteristics of the 1918 strain than a 1930s human virus. Based on the calculated rate of amino acid change in swine HA genes (30), one would expect approximately 20 changes between the 1918 strains and A/Swine/Iowa/30. We find 22 changes (of which 4 are in antigenic sites), consistent with the hypothesis that a virus similar to the 1918 strains entered the swine population in 1918 and changed gradually over the subsequent 12 years. By contrast, there are 43 amino acid differences between the 1918 strains and the human virus A/PR/8/34 (Cam), of which 15 are in antigenic sites, a rate of change consistent with a virus under substantial immune pressure.
Historical accounts document a widespread but mild wave of influenza in humans in the spring of 1918. Victims of the spring wave were reported to be immune from severe disease in the fall, suggesting that the spring and fall waves were closely related (10). Swine were first affected in the fall, when severe influenza-like disease outbreaks were noted in swine in the United States, Hungary, and China (34–36). Contemporary veterinarians had not seen this disease in pigs and believed it to be the same disease affecting humans. This sequence of events supports the theory that the 1918 influenza spread from humans to swine and is difficult to reconcile with the theory that separate human and swine H1 viruses were circulating from 1905 to 1918.
Analysis of the 1918 HA sequence permits alternative interpretations as to its origin. The 1918 sequences are phylogenetically distinct from current avian strains. One possibility is that around 1918 there existed an avian strain more similar to the pandemic virus than current avian strains and that its HA entered with little modification into the human population. If avian viruses have not drifted over the past 80 years, such a strain would differ from current avian strains phylogenetically. This hypothesis cannot be tested, because avian H1 influenza-virus isolates from that time do not exist. A second possibility is that the pandemic virus had been adapting in mammals before 1918 and that it had accumulated enough changes to make its HA gene seem more mammalian by many phylogenetic criteria (e.g., parsimony and NJ). Our data and those of others (29) suggest that an entry date into the human population between 1900 and 1915 is reasonable.
The 1918 influenza virus HA gene does not possess the cleavage site mutation seen in virulent avian influenza strains. No other known genetic changes were observed in the 1918 HA sequence that would account for the exceptional virulence of this pandemic virus. What determines the virulence of a particular influenza strain is quite complex and involves host adaptation, transmissibility, tissue tropism, and replication efficiency. The genetic basis for virulence of other influenza strains (for which complete genomic sequence is available) cannot be determined yet, but it is most likely polygenic in nature (4). Further sequence analyses of the remaining 1918 influenza genes are currently in progress, and it is hoped that these studies will shed additional light on the nature of the 1918 influenza virus.
J Gen Virol. 2003 Sep;84(Pt 9):2285-92.
The origin of the 1918 pandemic influenza virus: a continuing enigma.
Reid AH1, Taubenberger JK.
Author information 1Division of Molecular Pathology, Department of Cellular Pathology and Genetics, Armed Forces Institute of Pathology, 1413 Research Blvd, Building 101, Room 1057, Rockville, MD 20850-3125, USA.
Abstract Influenza A virus is a major public health threat, killing more than 30,000 per year in the USA alone, sickening millions and inflicting substantial economic costs. Novel influenza virus strains emerge periodically to which humans have little immunity, resulting in devastating pandemics. The 1918 pandemic killed nearly 700,000 Americans and 40 million people worldwide. Pandemics in 1957 and 1968, while much less devastating than 1918, also caused tens of thousands of deaths in the USA. The influenza A virus is capable of enormous genetic variability, both by continuous, gradual mutation and by reassortment of gene segments between viruses. Both the 1957 and 1968 pandemic strains are thought to have originated as reassortants, in which one or both human-adapted viral surface proteins were replaced by proteins from avian influenza virus strains. Analyses of the surface proteins of the 1918 pandemic strain, however, suggest that this strain may have had a different origin. The haemagglutinin gene segment of the virus may have come directly from an avian source different from those currently circulating. Alternatively, the virus, or some of its gene segments, may have evolved in an intermediate host before emerging as a human pathogen. Determining whether pandemic influenza virus strains can emerge via different pathways will affect the scope and focus of surveillance and prevention efforts. PMID: 12917448 DOI: 10.1099/vir.0.19302-0
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