Analysis by Single-Gene Reassortment Demonstrates that the 1918 Influenza Virus Is Functionally Compatible with a Low-Pathogenicity Avian Influenza Virus in Mice

The 1918-1919 "Spanish" influenza pandemic is estimated to have caused 50 million deaths worldwide. Understanding the origin, virulence, and pathogenic properties of past pandemic influenza viruses, including the 1918 virus, is crucial for current public health preparedness and future pandemic planning. The origin of the 1918 pandemic virus has not been resolved, but its coding sequences are very like those of avian influenza virus. The proteins encoded by the 1918 virus differ from typical low-pathogenicity avian influenza viruses at only a small number of amino acids in each open reading frame. In this study, a series of chimeric 1918 influenza viruses were created in which each of the eight 1918 pandemic virus gene segments was replaced individually with the corresponding gene segment of a prototypical low-pathogenicity avian influenza (LPAI) H1N1 virus in order to investigate functional compatibility of the 1918 virus genome with gene segments from an LPAI virus and to identify gene segments and mutations important for mammalian adaptation. This set of eight "7:1" chimeric viruses was compared to the parental 1918 and LPAI H1N1 viruses in intranasally infected mice. Seven of the 1918 LPAI 7:1 chimeric viruses replicated and caused disease equivalent to the fully reconstructed 1918 virus. Only the chimeric 1918 virus containing the avian influenza PB2 gene segment was attenuated in mice. This attenuation could be corrected by the single E627K amino acid change, further confirming the importance of this change in mammalian adaptation and mouse pathogenicity. While the mechanisms of influenza virus host switch, and particularly mammalian host adaptation are still only partly understood, these data suggest that the 1918 virus, whatever its origin, is very similar to avian influenza virus.     

1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds

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.     

Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus.

The Spanish influenza pandemic of 1918-1919 caused acute illness in 25-30% of the world's population and resulted in the death of 40 million people. The complete genomic sequence of the 1918 influenza virus will be deduced using fixed and frozen tissues of 1918 influenza victims. Sequence and phylogenetic analyses of the complete 1918 haemagglutinin (HA) and neuraminidase (NA) genes show them to be the most avian-like of mammalian sequences and support the hypothesis that the pandemic virus contained surface protein-encoding genes derived from an avian influenza strain and that the 1918 virus is very similar to the common ancestor of human and classical swine H1N1 influenza strains. Neither the 1918 HA genes nor the NA genes possessed mutations that are known to increase tissue tropicity, which accounts for the virulence of other influenza strains such as A/WSN/33 or fowl plague viruses. The complete sequence of the nonstructural (NS) gene segment of the 1918 virus was deduced and tested for the hypothesis that the enhanced virulence in 1918 could have been due to type I interferon inhibition by the NS1 protein. The results from these experiments were inconclusive. Sequence analysis of the 1918 pandemic influenza virus is allowing us to test hypotheses as to the origin and virulence of this strain. This information should help to elucidate how pandemic influenza strains emerge and what genetic features contribute to their virulence.     

Influenza A virus strains differ in sensitivity to the antiviral action of Mx-GTPase

Interferon-mediated host responses are of great importance for controlling influenza A virus infections. It is well established that the interferon-induced Mx proteins possess powerful antiviral activities toward most influenza viruses. Here we analyzed a range of influenza A virus strains for their sensitivities to murine Mx1 and human MxA proteins and found remarkable differences. Virus strains of avian origin were highly sensitive to Mx1, whereas strains of human origin showed much weaker responses. Artificial reassortments of the viral components in a minireplicon system identified the viral nucleoprotein as the main target structure of Mx1. Interestingly, the recently reconstructed 1918 H1N1 "Spanish flu" virus was much less sensitive than the highly pathogenic avian H5N1 strain A/Vietnam/1203/04 when tested in a minireplicon system. Importantly, the human 1918 virus-based minireplicon system was almost insensitive to inhibition by human MxA, whereas the avian influenza A virus H5N1-derived system was well controlled by MxA. These findings suggest that Mx proteins provide a formidable hurdle that hinders influenza A viruses of avian origin from crossing the species barrier to humans. They further imply that the observed insensitivity of the 1918 virus-based replicon to the antiviral activity of human MxA is a hitherto unrecognized characteristic of the "Spanish flu" virus that may contribute to the high virulence of this unusual pandemic strain.     

Preliminary Success in the Characterization and Management of a Sudden Breakout of a Novel H7N9 Influenza A Virus

Influenza has always been one of the major threats to human health. The Spanish influenza in 1918, the pandemic influenza A/H1N1 in 2009, and the avian influenza A/H5N1 have brought about great disasters or losses to mankind. More recently, a novel avian influenza A/H7N9 broke out in China and until December 2, 2013, it had caused 139 cases of infection, including 45 deaths. Its risk and pandemic potential attract worldwide attention. In this article, we summarize epidemiology, virology characteristics, clinical symptoms, diagnosis methods, clinical treatment and preventive measures about the avian influenza A/H7N9 virus infection to provide a reference for a possible next wave of flu outbreak.     

The genesis of a pandemic influenza virus

Pandemic influenza viruses pose a significant threat to public health worldwide. In a recent Nature paper, Taubenberger et al. (2005) now report remarkable similarities between the polymerase genes of the influenza virus that caused the 1918 Spanish influenza pandemic and those of avian influenza viruses. Meanwhile, Tumpey et al. (2005) reporting in Science show that the reconstructed 1918 Spanish influenza virus kills mice faster than any other influenza virus so far tested.     

The 1918-1919 influenza pandemic in England and Wales: spatial patterns in transmissibility and mortality impact

Spatial variations in disease patterns of the 1918-1919 influenza pandemic remain poorly studied. We explored the association between influenza death rates, transmissibility and several geographical and demographic indicators for the autumn and winter waves of the 1918-1919 pandemic in cities, towns and rural areas of England and Wales. Average measures of transmissibility, estimated by the reproduction number, ranged between 1.3 and 1.9, depending on model assumptions and pandemic wave and showed little spatial variation. Death rates varied markedly with urbanization, with 30-40% higher rates in cities and towns compared with rural areas. In addition, death rates varied with population size across rural settings, where low population areas fared worse. By contrast, we found no association between transmissibility, death rates and indicators of population density and residential crowding. Further studies of the geographical mortality patterns associated with the 1918-1919 influenza pandemic may be useful for pandemic planning.     

The anticipated severity of a "1918-like" influenza pandemic in contemporary populations: the contribution of antibacterial interventions

Recent studies have shown that most of deaths in the 1918 influenza pandemic were caused by secondary bacterial infections, primarily pneumococcal pneumonia. Given the availability of antibiotics and pneumococcal vaccination, how will contemporary populations fare when they are next confronted with pandemic influenza due to a virus with the transmissibility and virulence of that of 1918? To address this question we use a mathematical model and computer simulations. Our model considers the epidemiology of both the influenza virus and pneumonia-causing bacteria and allows for co-infection by these two agents as well as antibiotic treatment, prophylaxis and pneumococcal vaccination. For our simulations we use influenza transmission and virulence parameters estimated from 1918 pandemic data. We explore the anticipated rates of secondary pneumococcal pneumonia and death in populations with different prevalence of pneumococcal carriage and contributions of antibiotic prophylaxis, treatment, and vaccination to these rates. Our analysis predicts that in countries with lower prevalence of pneumococcal carriage and access to antibiotics and pneumococcal conjugate vaccines, there would substantially fewer deaths due to pneumonia in contemporary populations confronted with a 1918-like virus than that observed in the 1918. Our results also predict that if the pneumococcal carriage prevalence is less than 40%, the positive effects of antibiotic prophylaxis and treatment would be manifest primarily at of level of individuals. These antibiotic interventions would have little effect on the incidence of pneumonia in the population at large. We conclude with the recommendation that pandemic preparedness plans should consider co-infection with and the prevalence of carriage of pneumococci and other bacteria responsible for pneumonia. While antibiotics and vaccines will certainly reduce the rate of individual mortality, the factor contributing most to the relatively lower anticipated lethality of a pandemic with a 1918-like influenza virus in contemporary population is the lower prevalence of pneumococcal carriage.     

Origin of the 1918 pandemic H1N1 influenza A virus as studied by codon usage patterns and phylogenetic analysis

The pandemic of 1918 was caused by an H1N1 influenza A virus, which is a negative strand RNA virus; however, little is known about the nature of its direct ancestral strains. Here we applied a broad genetic and phylogenetic analysis of a wide range of influenza virus genes, in particular the PB1 gene, to gain information about the phylogenetic relatedness of the 1918 H1N1 virus. We compared the RNA genome of the 1918 strain to many other influenza strains of different origin by several means, including relative synonymous codon usage (RSCU), effective number of codons (ENC), and phylogenetic relationship. We found that the PB1 gene of the 1918 pandemic virus had ENC values similar to the H1N1 classical swine and human viruses, but different ENC values from avian as well as H2N2 and H3N2 human viruses. Also, according to the RSCU of the PB1 gene, the 1918 virus grouped with all human isolates and "classical" swine H1N1 viruses. The phylogenetic studies of all eight RNA gene segments of influenza A viruses may indicate that the 1918 pandemic strain originated from a H1N1 swine virus, which itself might be derived from a H1N1 avian precursor, which was separated from the bulk of other avian viruses in toto a long time ago. The high stability of the RSCU pattern of the PB1 gene indicated that the integrity of RNA structure is more important for influenza virus evolution than previously thought.     

The Leeuwenhoek Lecture 2001. Animal origins of human infectious disease

Since time immemorial animals have been a major source of human infectious disease. Certain infections like rabies are recognized as zoonoses caused in each case by direct animal-to-human transmission. Others like measles became independently sustained with the human population so that the causative virus has diverged from its animal progenitor. Recent examples of direct zoonoses are variant Creutzfeldt-Jakob disease arising from bovine spongiform encephalopathy, and the H5N1 avian influenza outbreak in Hong Kong. Epidemics of recent animal origin are the 1918-1919 influenza pandemic, and acquired immune deficiency syndrome caused by human immunodeficiency virus (HIV). Some retroviruses jump into and out of the chromosomal DNA of the host germline, so that they oscillate between being inherited Mendelian traits or infectious agents in different species. Will new procedures like animal-to-human transplants unleash further infections? Do microbes become more virulent upon cross-species transfer? Are animal microbes a threat as biological weapons? Will the vast reservoir of immunodeficient hosts due to the HIV pandemic provide conditions permissive for sporadic zoonoses to take off as human-to-human transmissible diseases? Do human infections now pose a threat to endangered primates? These questions are addressed in this lecture.     

The so-called Great Spanish Influenza Pandemic of 1918 may have originated in France in 1916.

This discussion piece examines the likely epicentre of the influenza pandemic of 1918-1919. Contrary to previous studies that have proposed a Chinese origin, there is documentation that suggests that, in this instance, the virus spread eastwards to China from Europe. Although more recent oubreaks of influenza have undoubtedly had an Oriental origin, the evidence indicates that future outbreaks could conceivably arise anywhere in the world.     

The predicted antigenicity of the haemagglutinin of the 1918 Spanish influenza pandemic suggests an avian origin.

In 1982 we characterized the antigenic sites of the haemagglutinin of influenza A/PR/8/34, which is an influenza strain of the H1 subtype that was isolated from humans in 1934, by studying mutants which escaped neutralization by antibody. Four antigenic sites, namely Cb, Sa, Sb and Ca, were found to be located near the tip of the trimeric haemagglutinin spike. Based on the sequence of the haemagglutinin of the 1918 Spanish influenza, we can now specify the extent of divergence of antigenic sites of the haemagglutinin during the antigenic drift of the virus between 1918 and 1934. This divergence was much more extensive (40%) than the divergence (20%) in predicted antigenic sites between the 1918 Spanish influenza and an avian H1 subtype consensus sequence. These results support the hypothesis that the human 1918 pandemic originated from an avian virus of the H1 subtype that crossed the species barrier from birds to humans and adapted to humans, presumably by mutation and/or reassortment, shortly before 1918.     

The beginning and ending of a respiratory viral pandemic-lessons from the Spanish flu

The COVID-19 pandemic goes into its third year and the world population is longing for an end to the pandemic. Computer simulations of the future development of the pandemic have wide error margins and predictions on the evolution of new viral variants of SARS-CoV-2 are uncertain. It is thus tempting to look into the development of historical viral respiratory pandemics for insight into the dynamic of pandemics. The Spanish flu pandemic of 1918 caused by the influenza virus H1N1 can here serve as a potential model case. Epidemiological observations on the shift of influenza mortality from very young and old subjects to high mortality in young adults delimitate the pandemic phase of the Spanish flu from 1918 to 1920. The identification and sequencing of the Spanish flu agent allowed following the H1N1 influenza virus after the acute pandemic phase. During the 1920s H1N1 influenza virus epidemics with substantial mortality were still observed. As late as 1951, H1N1 strains of high virulence evolved but remained geographically limited. Until 1957, the H1N1 virus evolved by accumulation of mutations (‘antigenic drift’) and some intratypic reassortment. H1N1 viruses were then replaced by the pandemic H2N2 influenza virus from 1957, which was in 1968 replaced by the pandemic H3N2 influenza virus; both viruses were descendants from the Spanish flu agent but showed the exchange of entire gene segments (‘antigenic shift’). In 1977, H1N1 reappeared from an unknown source but caused only mild disease. However, H1N1 achieved again circulation in the human population and is now together with the H3N2 influenza virus an agent of seasonal influenza winter epidemics.     

The low-pH stability discovered in neuraminidase of 1918 pandemic influenza A virus enhances virus replication

The "Spanish" pandemic influenza A virus, which killed more than 20 million worldwide in 1918-19, is one of the serious pathogens in recorded history. Characterization of the 1918 pandemic virus reconstructed by reverse genetics showed that PB1, hemagglutinin (HA), and neuraminidase (NA) genes contributed to the viral replication and virulence of the 1918 pandemic influenza virus. However, the function of the NA gene has remained unknown. Here we show that the avian-like low-pH stability of sialidase activity discovered in the 1918 pandemic virus NA contributes to the viral replication efficiency. We found that deletion of Thr at position 435 or deletion of Gly at position 455 in the 1918 pandemic virus NA was related to the low-pH stability of the sialidase activity in the 1918 pandemic virus NA by comparison with the sequences of other human N1 NAs and sialidase activity of chimeric constructs. Both amino acids were located in or near the amino acid resides that were important for stabilization of the native tetramer structure in a low-pH condition like the N2 NAs of pandemic viruses that emerged in 1957 and 1968. Two reverse-genetic viruses were generated from a genetic background of A/WSN/33 (H1N1) that included low-pH-unstable N1 NA from A/USSR/92/77 (H1N1) and its counterpart N1 NA in which sialidase activity was converted to a low-pH-stable property by a deletion and substitutions of two amino acid residues at position 435 and 455 related to the low-pH stability of the sialidase activity in 1918 NA. The mutant virus that included "Spanish Flu"-like low-pH-stable NA showed remarkable replication in comparison with the mutant virus that included low-pH-unstable N1 NA. Our results suggest that the avian-like low-pH stability of sialidase activity in the 1918 pandemic virus NA contributes to the viral replication efficiency.     

Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus

Annual outbreaks of influenza A infection are an ongoing public health threat and novel influenza strains can periodically emerge to which humans have little immunity, resulting in devastating pandemics. The 1918 pandemic killed at least 40 million people worldwide and pandemics in 1957 and 1968 caused hundreds of thousands of deaths. The influenza A virus is capable of enormous genetic variation, both by continuous, gradual mutation and by reassortment of genome 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 strains. Analyses of the genes of the 1918 pandemic virus, however, indicate that this strain might have had a different origin. The haemagglutinin and nucleoprotein genome segments in particular are unlikely to have come directly from an avian source that is similar to those that are currently being sequenced. Determining whether a pandemic influenza virus can emerge by different mechanisms will affect the scope and focus of surveillance and prevention efforts.     

Pathogenicity of influenza viruses with genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in mice

The Spanish influenza pandemic of 1918 to 1919 swept the globe and resulted in the deaths of at least 20 million people. The basis of the pulmonary damage and high lethality caused by the 1918 H1N1 influenza virus remains largely unknown. Recombinant influenza viruses bearing the 1918 influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins were rescued in the genetic background of the human A/Texas/36/91 (H1N1) (1918 HA/NA:Tx/91) virus. Pathogenesis experiments revealed that the 1918 HA/NA:Tx/91 virus was lethal for BALB/c mice without the prior adaptation that is usually required for human influenza A H1N1 viruses. The increased mortality of 1918 HA/NA:Tx/91-infected mice was accompanied by (i) increased (>200-fold) viral replication, (ii) greater influx of neutrophils into the lung, (iii) increased numbers of alveolar macrophages (AMs), and (iv) increased protein expression of cytokines and chemokines in lung tissues compared with the levels seen for control Tx/91 virus-infected mice. Because pathological changes in AMs and neutrophil migration correlated with lung inflammation, we assessed the role of these cells in the pathogenesis associated with 1918 HA/NA:Tx/91 virus infection. Neutrophil and/or AM depletion initiated 3 or 5 days after infection did not have a significant effect on the disease outcome following a lethal 1918 HA/NA:Tx/91 virus infection. By contrast, depletion of these cells before a sublethal infection with 1918 HA/NA:Tx/91 virus resulted in uncontrolled virus growth and mortality in mice. In addition, neutrophil and/or AM depletion was associated with decreased expression of cytokines and chemokines. These results indicate that a human influenza H1N1 virus possessing the 1918 HA and NA glycoproteins can induce severe lung inflammation consisting of AMs and neutrophils, which play a role in controlling the replication and spread of 1918 HA/NA:Tx/91 virus after intranasal infection of mice.     

Corporate preparedness for pandemic influenza: a survey of pharmaceutical and biotechnology companies in Montgomery County, Maryland

Objectives: We conducted a survey of corporate preparedness for pandemic influenza among biotechnology and pharmaceutical companies in Montgomery County, Maryland, to determine the level of preparedness for this industry and geographic region. Methods: The survey, based on the HHS Business Pandemic Influenza Planning Checklist, established whether a company had a preparedness plan specific to pandemic influenza, the contents of its plan, or its reasons for a lack of a plan. Results: A total of 50 companies participated in the survey. Of these, 40 did not have any type of preparedness plan, 3 were drafting plans, 6 had general preparedness plans that could be applied to an influenza pandemic, and only 1 company had a preparedness plan specifically designed to address pandemic influenza. Conclusions: Biotechnology and pharmaceutical companies in this geographic region are currently not well prepared for pandemic influenza. Public health officials should offer more help, possibly in the form of a model small business preparedness plan, and collaboration between companies should be encouraged to foster sharing of preparedness plans.     

Elicitation of anti-1918 influenza virus immunity early in life prevents morbidity and lower levels of lung infection by 2009 pandemic H1N1 influenza virus in aged mice

The Spanish influenza virus pandemic of 1918 was responsible for 40 million to 50 million deaths and is antigenically similar to the swine lineage 2009 pandemic influenza virus. Emergence of the 2009 pandemic from swine into humans has raised the possibility that low levels of cross-protective immunity to past shared epitopes could confer protection. In this study, influenza viruslike particles (VLPs) were engineered to express the hemagglutinin (HA) and genes from the 1918 influenza virus to evaluate the duration of cross-protection to the H1N1 pandemic strain by vaccinating young mice (8 to 12 weeks) and then allowing the animals to age to 20 months. This immunity was long lasting, with homologous receptor-blocking antibodies detected throughout the lifespan of vaccinated mice. Furthermore, the 1918 VLPs fully protected aged mice from 2009 pandemic H1N1 virus challenge 16 months after vaccination. Histopathological assessment showed that aged vaccinated mice had significant protection from alveolar infection but less protection of the bronchial tissue than adult vaccinated mice. Additionally, passive transfer of immune serum from aged vaccinated mice resulted in protection from death but not morbidity. This is the first report describing the lifelong duration of cross-reactive immune responses elicited by a 1918 VLP vaccine in a murine model. Importantly, these lifelong immune responses did not result in decreased total viral replication but did prevent infection of the lower respiratory tract. These findings show that immunity acquired early in life can restrict the anatomical location of influenza viral replication, rather than preventing infection, in the aged.     

Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 2007¹ and resulted in the hospitalization of 1.1 million patients². With an average length of in-patient hospital stay of five days², pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 2005³. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence⁴. (Figure 1)Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients’ length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone⁵. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians⁶. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems^{7,8, 9, 10}.The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.^5,11