Thomas A. Janczewski

Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes

The influenza A virus pandemic of 1918–1919 resulted in an estimated 20–40 million deaths worldwide. The hemagglutinin and neuraminidase sequences of the 1918 virus were previously determined. We here report the sequence of the A/Brevig Mission/1/18 (H1N1) virus nonstructural (NS) segment encoding two proteins, NS1 and nuclear export protein. Phylogenetically, these genes appear to be close to the common ancestor of subsequent human and classical swine strain NS genes. Recently, the influenza A virus NS1 protein was shown to be a type I IFN antagonist that plays an important role in viral pathogenesis. By using the recently developed technique of generating influenza A viruses entirely from cloned cDNAs, the hypothesis that the 1918 virus NS1 gene played a role in virulence was tested in a mouse model. In a BSL3+ laboratory, viruses were generated that possessed either the 1918 NS1 gene alone or the entire 1918 NS segment in a background of influenza A/WSN/33 (H1N1), a mouse-adapted virus derived from a human influenza strain first isolated in 1933. These 1918 NS viruses replicated well in tissue culture but were attenuated in mice as compared with the isogenic control viruses. This attenuation in mice may be related to the human origin of the 1918 NS1 gene. These results suggest that interaction of the NS1 protein with host-cell factors plays a significant role in viral pathogenesis.

1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants.

The “Spanish influenza pandemic swept the globe in the autumn and winter of 1918–19, and resulted in the deaths of approximately 40 million people. Clinically, epidemiologically, and pathologically, the disease was remarkably uniform, which suggests that similar viruses were causing disease around the world. To assess the homogeneity of the 1918 pandemic influenza virus, partial hemagglutinin gene sequences have been determined for five cases, including two newly identified samples from London, United Kingdom. The strains show 98.9% to 99.8% nucleotide sequence identity. One of the few differences between the strains maps to the receptor-binding site of hemagglutinin, suggesting that two receptor-binding configurations were co-circulating during the pandemic. The results suggest that in the early stages of an influenza A pandemic, mutations that occur during replication do not become fixed so that a uniform “consensus” strain circulates for some time.

Novel Origin of the 1918 Pandemic Influenza Virus Nucleoprotein Gene

ABSTRACT The nucleoprotein (NP) gene of the 1918 pandemic influenza A virus has been amplified and sequenced from archival material. The NP gene is known to be involved in many aspects of viral function and to interact with host proteins, thereby playing a role in host specificity. The 1918 NP amino acid sequence differs at only six amino acids from avian consensus sequences, consistent with reassortment from an avian source shortly before 1918. However, the nucleotide sequence of the 1918 NP gene has more than 170 differences from avian strain consensus sequences, suggesting substantial evolutionary distance from known avian strain sequences. Both the gene and protein sequences of the 1918 NP fall within the mammalian clade upon phylogenetic analysis. The evolutionary distance of the 1918 NP sequences from avian and mammalian strain sequences is examined, using several different parameters. The results suggest that the 1918 strain did not retain the previously circulating human NP. Nor is it likely to have obtained its NP by reassortment with an avian strain similar to those now characterized. The results are consistent with the existence of a currently unknown host for influenza, with an NP similar to current avian strain NPs at the amino acid level but with many synonymous nucleotide differences, suggesting evolutionary isolation from the currently characterized avian influenza virus gene pool.

Characterization of the 1918 “Spanish” Influenza Virus Matrix Gene Segment

ABSTRACT The coding region of influenza A virus RNA segment 7 from the 1918 pandemic virus, consisting of the open reading frames of the two matrix genes M1 and M2, has been sequenced. While this segment is highly conserved among influenza virus strains, the 1918 sequence does not match any previously sequenced influenza virus strains. The 1918 sequence matches the consensus over the M1 RNA-binding domains and nuclear localization signal and the highly conserved transmembrane domain of M2. Amino acid changes that correlate with high yield and pathogenicity in animal models were not found in the 1918 strain. Phylogenetic analyses suggest that both genes were mammalian adapted and that the 1918 sequence is very similar to the common ancestor of all subsequent human and classical swine matrix segments. The 1918 sequence matches other mammalian strains at 4 amino acids in the extracellular domain of M2 that differ consistently between avian and mammalian strains, suggesting that the matrix segment may have been circulating in human strains for at least several years before 1918.

1917 Avian Influenza Virus Sequences Suggest that the 1918 Pandemic Virus Did Not Acquire Its Hemagglutinin Directly from Birds

ABSTRACT Wild waterfowl captured between 1915 and 1919 were tested for influenza A virus RNA. One bird, captured in 1917, was infected with a virus of the same hemagglutinin (HA) subtype as that of the 1918 pandemic virus. The 1917 HA is more closely related to that of modern avian viruses than it is to that of the pandemic virus, suggesting (i) that there was little drift in avian sequences over the past 85 years and (ii) that the 1918 pandemic virus did not acquire its HA directly from a bird.

Age- and Sex-Specific Mortality Associated With the 1918–1919 Influenza Pandemic in Kentucky

Abstract Background. The reasons for the unusual age-specific mortality patterns of the 1918–1919 influenza pandemic remain unknown. Here we characterize pandemic-related mortality by single year of age in a unique statewide Kentucky data set and explore breakpoints in the age curves. Methods. Individual death certificates from Kentucky during 1911–1919 were abstracted by medically trained personnel. Pandemic-associated excess mortality rates were calculated by subtracting observed rates during pandemic months from rates in previous years, separately for each single year of age and by sex. Results. The age profile of excess mortality risk in fall 1918 was characterized by a maximum among infants, a minimum at ages 9–10 years, a maximum at ages 24–26 years, and a second minimum at ages 56–59 years. The excess mortality risk in young adults had been greatly attenuated by winter 1919. The age breakpoints of mortality risk did not differ between males and females. Conclusions. The observed mortality breakpoints in male and female cohorts born during 1859–1862, 1892–1894, and 1908–1909 did not coincide with known dates of historical pandemics. The atypical age mortality patterns of the 1918–1919 pandemic cannot be explained by military crowding, war-related factors, or prior immunity alone and likely result from a combination of unknown factors.

Reply to Wilson et al

To the Editor— We thank Wilson et al [1] for their comments on our study of the 1918–1919 influenza pandemic in Kentucky [2]. Our key findings were to provide evidence of break points in the mortality profile of the fall 1918 pandemic wave among individuals aged 9–10 years (ie, cohorts born during 1908–1909) and those aged 24–26 years (ie, cohorts born during 1892–1894) [2]. These age break points do not strictly align with known dates of past pandemic events, and, hence, it is not intuitive that they support or refute the hypothesis proposed by Wilson et al, in which aberrant immune responses mediated by CD8+ T cells were more frequent among young adults whose first influenza virus exposures were to the 1889 pandemic virus [1]. In their letter, Wilson et al contribute interesting data on influenza mortality patterns in New Zealand [1]. They report a mortality peak at ages 29–30 years in males and females (ie, cohorts born during 1888–1889), which is more in line with hypotheses invoking the 1889 pandemic than our Kentucky data. These findings could reflect true differences in the pandemic experience of geographically distant populations. Alternatively, the mortality profile in New Zealand could be influenced by a preponderance of influenza risk factors in aboriginal Maori populations or by high variance in mortality estimates due to small population size (approximately 0.85 million). Methodological factors may also contribute to the observed differences between the 2 studies, as our Kentucky analysis relied on “above baseline” excess mortality in the lethal months of October–December 1918 [2]. Future studies could compare the risks profiles of different populations via the excess mortality approach to help tease out influenza-specific effects from unrelated background mortality [3]. Wilson et al note that US population size estimates from this period may be inaccurate, potentially resulting in imprecise death rate estimates—an issue emphasized by an earlier historical study from Canada and the United States [4]. We agree that estimates of male population sizes during wartime are prone to bias because of troop movements. Hence, like Wilson et al, we combined female mortality counts with population information collated from carefully conducted decennial censuses to present a relatively unbiased picture of mortality risk at the time. We have now repeated our analysis with death count data, which we show in parallel with our analysis of excess death rates in Figure ​Figure1.1. Both curves indicate mortality peaks at ages 25–26 years (ie, cohorts born during 1892–1893), consistent across various diagnostic outcomes and in female-specific data. These sensitivity analyses confirm that the Kentucky cohorts at highest risk of influenza-related death were those born a few years after the 1889 pandemic, irrespective of whether we use population denominators. Figure 1. Age-specific patterns of mortality due to respiratory disease during the October–December 1918 influenza pandemic wave in Kentucky. A, Data obtained using a regression model to estimate the influenza-related mortality rate (ie, the excess mortality ... Overall, our historical analysis provides unprecedented detail on the age and sex patterns of the pandemic in Kentucky [2]. Similar to Wilson et al [1], we found a substantially increased risk of death among military populations, perhaps due to crowding and/or increased circulation of coinfecting pathogenic bacteria. However, these factors alone cannot account for the mortality risk of the 1918 pandemic among young adult civilian females. While the reasons for the unusual age patterns of the 1918 pandemic are difficult to elucidate from epidemiological data alone, the steep rise in the risk of mortality among individuals aged 10–20 years during the influenza pandemic is worth noting, especially as it consistent in data from New Zealand, Canada, and the United States [1, 2, 4]. Historical morbidity surveys indicate that clinical attack rates were similar in these age cohorts [5], suggesting that the severity of influenza-related infection increased sharply between ages 10 and 20 years, likely because of a heightened risk of pneumonia caused by common bacterial respiratory pathogens (especially pneumococci, streptococci, and staphylococci) [5–7]. People aged 10–20 years had not lived through the 1889 pandemic, although they could have been infected by descendants of the 1889 influenza virus that persisted during 1892–1918. Although the reasons for the atypical mortality risk profile of the 1918–1919 pandemic may remain elusive, it is important to pursue efforts to analyze archival mortality records from a variety of locations, building upon the work by Wilson et al and others [1, 2, 4, 5, 8, 9]. A systematic epidemiological description of the pandemic in a variety of globally sampled populations may provide unique insights into the host and geographic factors responsible for the unusual severity of disease associated with the 1918 pandemic virus.

Seeking a Pale Horse: The 1918 Pandemic Influenza Virus

Influenza A viruses are negative strand RNA viruses. Their segmented genome consists of eight RNA segments coding for ten proteins. Two glycosylated proteins, hemagglutinin (HA) and neuraminidase (NA), are involved in viral attachment to cells. Two nonstructural proteins, NS1 and NS2 (also called NEP), are involved in regulating numerous aspects of the viral life cycle. These four proteins will be discussed in more detail below. Three viral proteins, PA, PB1 and PB2, are responsible for viral replication, while the nucleoprotein, NP, is the nucleocapsid structural protein. Finally, two membrane proteins, Ml and M2, appear to be involved in nuclear export and pH maintenance, among other activities (Lamb and Krug, 1996).

Characterization of the 1918 influenza virus hemagglutinin and neuraminidase genes

Abstract In the fall and winter of 1918–1919, an influenza pandemic of unprecedented virulence swept the globe leaving 40 million or more dead in its wake. The virus responsible for this catastrophe was not isolated at the time, however, it has recently become possible to study the genetic features of the 1918 ‘Spanish’ influenza virus using frozen and fixed autopsy tissue. Gene sequences of the 1918 virus can be used to frame hypotheses about the origin of the 1918 virus, and to look for clues to its virulence. The study of the 1918 virus is not just one of historical curiosity. An understanding of the genetic make-up of the most virulent influenza strain in history may facilitate prediction and prevention of future pandemics.