Identifying and Characterizing the Causative Agent

Given the prominence of the influenza virus in human history – ranging from seasonal outbreaks to devastating pandemics such as the 1918 H1N1 flu – it is not surprising that there is abundance of literature attempting to elucidate and understand the causative agent of the disease. Through clinical (or general) observation, it was clear that this disease was infectious by nature, but once available technologies permitted a more definitive investigation, there was some controversy surrounding the causative pathogen.4 

In 1892, Richard Pfeiffer identified a bacillus bacterium common to nasal secretions of many influenza patients, which he assumed to be the causative agent of infection. The bacterium, B. influenzae, was termed “Pfeiffer’s bacillus,” and generally accepted as the cause of influenza infection for nearly two decades to follow.1,2 The severity of the 1918 influenza pandemic, though, prompted further study.1 In the same year that Pfeiffer identified B. influenzae, botanist Dmitri Ivanoski had reported discovery of a novel “filterable pathogen” plaguing tobacco plants – the adjective referring to the presence of the pathogen in filtrate from Chamerland filter-candles, which were known to catch bacteria. In 1898, this pathogen was termed a “virus.”12

In the 1930s, following the shock of the 1918 outbreak, Richard Shope attempted to identify the infectious cause of an influenza-like illness in pigs. Though he initially isolated a bacterium consistent with the bacillus identified by Pfeiffer, he found that this microbe did not cause disease when injected into healthy pigs.1 Shope attempted to filter the pathogen, as Ivanoski had done with the infection from his tobacco plants, and found that the infectious agent was indeed present in the filtrate.1 In 1933, the influenza A virus was successfully isolated for the first time, a landmark finding which corroborated Shope’s work and propelled influenza research dramatically.1

Following identification of the pathogen, literature in this area shifted from investigating what was causing the disease and instead began to focus on characterizing the virus. Influenza viruses are broken down into four classes: Influenza A and Influenza B, as well as Influenza C and D, though A and B are the primary viruses implicated in causing human disease.5 Influenza A viruses are classified into subtypes according to their membrane haemagglutinin and neuraminidase protein component types (Fig. 1), signified by the H/N nomenclature frequently used to describe seasonal influenzas.5 Though most often (currently and historically) identified antigenically,these proteins can also be classified phylogenetically.7 Influenza A H1N1 is one of the most historically relevant and dangerous flu strains, causing both the 1918 Spanish Flu4 and the 2009 Swine Flu epidemics.2 Influenza subtypes can be further classified into strains based on more discrete genetic differences.5 These strains are typically named according to their subtype and the place in which the virus is initially identified.5 

Figure 1. A depiction of the influenza A virus, highlighting the hemagglutinin and neuraminidase surface proteins, as well as the M2 channel and core RNPs (NPs bound vRNAs).13

Like all viruses, influenza contains the genetic material necessary to replicate and build its protein coat, but must exploit the machinery of a host cell during infection to synthesize new viruses, as it lacks components essential to doing so on its own.5 Influenza viruses are part of the Orthomyxoviridae family, and all are negative-sense RNA enveloped viruses.5 Influenza A carries 8 modular single stranded RNA segments which encode proteins necessary for viral replication and function. The major proteins encoded by these RNA segments are PB1, PB2, PA, HA, NP, NA, M (M1 and M2), and NS (NS1 and NEP). PB1, PB2, and PA are the three subunits which make up the viral RNA dependent polymerase essential for viral replication during infection. HA refers to the aforementioned hemagglutinin protein, a surface protein on the viral envelope which mediates viral entry to host cells, and NA refers to the neuraminidase enzyme responsible for mediating viral release. The nucleoprotein, NP, interacts with viral RNA and the polymerase components to form ribonucleoprotein complexes (RNPs). The matrix, M1, and membrane ion channel, M2, proteins are involved in viral structure and viral entry and exit respectively. Finally, the NS1 (non-structural protein 1) protein counteracts host immune responses.5 Genetic differences in these proteins are often what determine strain classifications.5

As evident in the history of its identification, influenza viruses are found not only in humans, but in a variety of animal hosts as well.5 Typically, influenza A viruses will circulate in animal populations before passing to a human host. This can occur when an individual consumes infected, uncooked meat, or is otherwise in close contact with an infected animal.5

One of the most fascinating and troubling characteristics of the influenza virus is its capacity for change. When two different influenza A subtypes infect the same cell, these modular RNA components can recombine to generate new subtype combinations, a process known as antigenic shift. Additionally, the viral genome of influenza is extremely prone to mutation, known as antigenic drift, as diagrammed below.5 Both of these processes contribute to the frequent changes circulating influenza viruses. This is the primary contributing factor to the high virulence of influenza year to year, particularly when the virus is antigenically novel and there is little herd immunity, which has also led to difficulties in vaccine efficacy.5

Figure 2. Visual representation of genetic changes in influenza viruses. This ability is a significant contributing factor to the historical severity of influenza outbreaks.5

While H1N1 has been the source of two of the most prominent influenza pandemics of the past century, it is not the only influenza subtype capable of causing such devastating disease. Recently, public health officials have expressed concern about the prospect of an H5N1 pandemic, as this virus has shown to be severely pathogenic. Currently, though, circulating strains of H5N1 have had very poor human to human transmission.5 It has been demonstrated that only minor changes to the H5N1 virus are necessary for transmission in ferrets, speaking to the profound epidemiological impact such genetic changes can have.5 

In combating these prospective pandemics, though, understanding beyond the general characterization presented here is necessary. The following pages will discuss the molecular research which has informed current influenza understanding and therapeutic approaches.

Summary of Major Scientific Innovations:

  • 1892 – Pfiefer’s bacteria (“bacillus of influenza”) is identified as causative agent 1,2
    Pfiefer identified a bacillus bacteria in the respiratory tract of patients presenting with influenza-like symptoms. This was thought to be the causative agent for some time, before subsequent work suggested it may be viral. Some early attempts at vaccine development were actually bacterial. 
  • 1892 – A “filterable pathogen” is identified in by Russian botanist Ivanoski12
    Today, we know this to be the Tobacco mosaic virus – the first virus discovered.
  • 1898 – The term “virus” is first used to describe the Tobacco pathogen12
  • 1918 – Gibson and Connor demonstrate the influenza from the 1918 outbreak is caused by a filterable virus 3
    Gibson and Connor demonstrate that the causative agent for influenza is a filterable virus during the 1918 pandemic, confirming findings of Schope, appearing to shift the literature away from Pfieffer’s bacteria. 
  • 1933 – The influenza virus is isolated 4
  • 1952 – Demonstrated that influenza can genetically recombine 8
  • 1976-1977 – Mapping of the influenza genome 9,10,11
    Identifies genes encoding the primary viral proteins necessary for infection and viral assembly 

Works Cited

  1. Van Epps, H.L. Influenza: exposing the true killer. J Exp Med. 2006, 203(4): 803. 
  2. Robertson, W.F. Influenza: Its Cause and Prevention. Br Med J. 1918, 2(3025):680-681. 
  3. Gibson, H.G., and Connor, J.I. A filterable virus as the cause of the early stage of the present epidemic influenza. Br Med J. 1918, 2(3024):645-646. 
  4. Smith, W., Andrewes, C.H., and Laidlaw, P.P. [Reviewed] Timbury, M.C. A Virus obtained from influenza Patients. Reviews in Medical Virology. 1995* (1933), 5:187-191
  5. Krammer, F.; Smith, G. J. D.; Fouchier, R. A. M.; Peiris, M.; Kedzierska, K.; Doherty, P. C.; Palese, P.; Shaw, M. L.; Treanor, J.; Webster, R. G.; et al. Influenza. Nat. Rev. Dis. Primer 2018, 4 (1), 1–21.
  6. Taubenberger, J.K. and Morens, D.M. The Pathology of Influenza Virus Infections. Annu Rev Pathol 2008 3:499-522.
  7. Sutton, T.C.; Chakraborty, S.; Mallajosyula, V.A.; Lamirande, E.W.; Gganti, K.; Bock, K.W.; Moore, I.N.; Varadarajan, R.; and Subbarao, K. Protective Efficacy of Influenza Group 2 Hemagglutinin Stem-Fragment Immunogen Vaccines. NPJ Vaccines 2017 2:35.
  8. Burnet, F.M.; and Lind, P.E. A genetic approach to variation in influenza viruses; recombination of characters between the influenza virus A strain NWS and strains of different serological types. J Gen Microbiol. 1951, 5(1):67-82. 
  9. Palese, P., and Schulman J.L. Mapping of the influenza virus genome: identification of the hemagglutinin and the neuraminidase genes. Proc Natl Acad Sci USA. 1976, 73(6):2142-46.
  10. Palese, P., Ritchey, M.B., and Schulman J.L. Mapping of the influenza virus genome II: identification of the P1, P2, and P3 genes. Proc Natl Acad Sci USA. 1977, 76(1):114-121
  11. Ritchey, M.B., Palese, P., and Schulman J.L. Mapping of the influenza virus genome III: identification of genes coding for nucleoprotein, membrane protein, and nonstructural protein. Journal of Virology. 1976, 20(1):307-313
  12. Lecoq, H. Discovery of the First Virus, the Tobacco Mosaic Virus: 1892 or 1898? C R Acad Sci III. 2001, 324(10):929-933.
  13. CDC. Types of Influenza Viruses. (Accessed March 12, 2020).

For more information and resources, see Annotated Bibliography.

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