Abstract

Influenza-related pathologies affect millions of people each year and the impact of influenza on the global economy and in our everyday lives has been well documented. Influenza viruses not only infect humans but also are zoonotic pathogens that infect various avian and mammalian species, which serve as viral reservoirs. While there are several strains of influenza currently circulating in animal species, H2 influenza viruses have a unique history and are of particular concern. The 1957 ‘Asian Flu’ pandemic was caused by H2N2 influenza viruses and circulated among humans from 1957 to 1968 before it was replaced by viruses of the H3N2 subtype. This review focuses on avian influenza viruses of the H2 subtype and the role these viruses play in human infections. H2 influenza viral infections in humans would present a unique challenge to medical and scientific researchers. Much of the world's population lacks any pre-existing immunity to the H2N2 viruses that circulated 50–60 years ago. If viruses of this subtype began circulating in the human population again, the majority of people alive today would have no immunity to H2 influenza viruses. Since H2N2 influenza viruses have effectively circulated in people in the past, there is a need for additional research to characterize currently circulating H2 influenza viruses. There is also a need to stockpile vaccines that are effective against both historical H2 laboratory isolates and H2 viruses currently circulating in birds to protect against a future pandemic.

Introduction

Influenza A viruses are part of the Orthomyxoviridae family and the genome consists of eight negative sense RNA strands that encode for 12 different proteins, which include hemagglutinin (HA), neuraminidase (NA), nonstructural protein 1 (NSP1), nonstructural protein 2 (NSP2), matrix 1 (M1), matrix 2 (M2), nucleoprotein (NP), nuclear export protein (NEP), polymerase basic protein (PA), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2) and polymerase basic protein 1–F2 (PB1-F2) [1]. The major viral receptor glycoprotein on the surface of the virus, HA, mediates host–receptor binding and entry into cells [2], (Figure 1). HA binds to sialic acid, a common receptor on mammal and avian cells. The second major glycoprotein, NA, mediates viral egress and nascent viral escape from infected cells. Influenza viruses are classified based on both the sequence and antigenic differences of the two surface glycoproteins, HA and NA. For example, the 2009 influenza pandemic contained a hemagglutinin type 1 and neuraminidase type 1 and these viruses were designated as an H1N1 subtype. There are 18 different HA proteins and 11 different NA proteins that result in a variety of influenza subtypes [2].

Influenza A virus structure and genome.

Figure 1.
Influenza A virus structure and genome.

Trimeric hemagglutinin glycoprotein is shown in red. Tetrameric neuraminidase glycoprotein is shown in blue. Membrane Protein 1 (M1) is shown in blue in the inner membrane. Membrane Protein 2 (M2) is shown bridging the viral shell in orange. The nuclear export protein is shown inside the viral vesicle in yellow. Nucleocapsid Protein (NP) is shown in light brown. Each of the eight viral RNA genomic segments is shown as red lines coiled around the NP proteins with each segment labeled. The RNA polymerase complex consisting of PB1, PB2, and PA is shown as the blue, green, and turquoise circle bunched together at the end of each viral genome segment [1].

Figure 1.
Influenza A virus structure and genome.

Trimeric hemagglutinin glycoprotein is shown in red. Tetrameric neuraminidase glycoprotein is shown in blue. Membrane Protein 1 (M1) is shown in blue in the inner membrane. Membrane Protein 2 (M2) is shown bridging the viral shell in orange. The nuclear export protein is shown inside the viral vesicle in yellow. Nucleocapsid Protein (NP) is shown in light brown. Each of the eight viral RNA genomic segments is shown as red lines coiled around the NP proteins with each segment labeled. The RNA polymerase complex consisting of PB1, PB2, and PA is shown as the blue, green, and turquoise circle bunched together at the end of each viral genome segment [1].

Humans have been infected with influenza viruses containing hemagglutinin type H1, H2, or H3 with neuraminidases N1 or N2. While influenza viruses can infect a multitude of different animal species, only influenza viruses with hemagglutinin types H1, H2, or H3 have been shown to efficiently transmit between people [1,2]. However, influenza viruses classified in the subtypes H5, H7, H9, and H10 have infected humans directly from zoonotic reservoirs without demonstrating the ability to efficiently transmit between humans. While these viruses readily infect birds, they do not efficiency transmit between humans [3]. In this article, the pathogenesis, evolution, and potential for H2 influenza viruses to cause a pandemic is reviewed.

Wild birds and influenza surveillance

Avian species, particularly waterfowl, shorebirds, and seabirds, are an important reservoir for influenza viruses with more than 100 varieties of birds with recorded infections. The prevalence of influenza viruses isolated from waterfowl is most likely due to the congregation of numbers of birds in and around wetlands. This congregation is likely also responsible for the vast diversity of influenza combinations in avian species. [4]. Influenza viruses can survive in lakes for up to 4 days at a temperature of 22°C and at least 30 days when the temperature is below 0°C [5]. The ability of influenza viruses to survive freezing temperatures enables the viral transmission between birds that are migrating to warmer climates in the fall to birds that are returning to the more temperate climates in the spring [6].

Avian influenza strains such as H5, H7, and H9 are most commonly low-pathogenic in birds. However, some strains of H5 and H7 are highly pathogenic to Gallus birds, such as chickens and turkeys. While influenza viruses efficiently spread among birds, the ability of these viruses to directly infect humans is not clear due to a multitude of ecological factors and the addition of intermediate animal hosts that makes modeling avian influenza virus transmission to humans extremely difficult [7]. However, there is evidence to suggest that migrating birds were responsible for the spread of H5N1 viruses among domestic birds throughout Eurasia [8].

Surveillance of influenza in avian species is conducted on a constant basis [9]. Most avian influenza surveillance programs are searching for HPAI, such as H5 or H7 strains. However, LPAI strains are routinely found during surveillance [10]. A comprehensive review of 191 published surveillance reports of wild avian species was conducted in 2010 [10]. H2 was isolated in several of the reports encompassed in this study. Additionally, H2 has been isolated in several more recent reports. H2 viruses were isolated from ducks in Alberta, Canada from 2001 to 2006 [11]. H2N5 influenza viruses were isolated from Heron Island, Australia in 2004 [12]. A multiyear surveillance of poultry in Belgium conducted from 2007 to 2013 revealed the presence of several H2 viruses [13]. In 2011, an H2N7 virus was isolated from a ruddy turnstone in Iceland [9]. H2N7 and H2N8 viruses were identified in 2012 and 2013, respectively in live poultry markets in China [14,15]. Another study analyzed the phylogenetics of several recent H2 isolated and concluded that there is an intercontinental exchange of H2 strains between North America and Eurasian lineages [16]. These findings indicate that H2 is still actively circulating in avian species, albeit less than some other influenza subtypes.

Poultry

H2 influenza viruses have been isolated from poultry on a few occasions in the United States and Europe [13,17]. These viruses have all had low pathogenicity in poultry. Low pathogenicity avian influenza (LPAI) viruses typically elicit symptoms such as sneezing, coughing, and nasal discharge in poultry and sinusitis in waterfowl and quail [18]. Morbidity and mortality rates are usually low for LPAI viruses with H9N2 viral isolates being the most common LPAI virus throughout most of the world [18]. High pathogenicity avian influenza (HPAI) viruses usually cause severe disease with mortality rates near 100%. The progress of HPAI disease can be rapid in poultry. Acute infections result in lesions, edema, and subcutaneous hemorrhaging. From the time of onset of symptoms, death can occur in less than 2 days. While not all birds survive infection, those that do survive can develop neurological symptoms, such as paralysis and drooping wings. Interactions with dead birds or live birds infected with LPAI and HPAI viruses are the main sources of infection for humans. Depending on the specific virus, LPAI and HPAI elicit symptoms in humans ranging from mild respiratory distress to severe lower respiratory infections and even death [18].

Multibasic cleavage sites

Multibasic cleavage sites are directly related to the pathogenicity of influenza viruses in poultry. When an influenza virus attaches to a cell, the HA protein is in an uncleaved form called HA0. When the virus begins to enter the cell, the HA protein undergoes a cleavage that separates the HA protein into two functional parts called HA1 and HA2. The HA1 portion of the HA protein stays bound to the target cell's sialic acid. The HA2 portion of the HA protein contains the fusion peptide that is responsible for fusing the viral membrane with the endosomal membrane which releases the viral RNA into the target cell [19]. LPAI viruses have an arginine residue at the cleavage site between HA1 and HA2 and are cleaved by trypsin-like proteases [20]. These trypsin-like proteases are found in the respiratory and gastrointestinal tract of poultry that restricts influenza virus replication to those areas. Alternatively, multibasic cleavage sites are cleaved by furin-like proteases. The multibasic cleavage sites are created by the addition of basic amino acids. These basic amino acids are most likely the result of ‘slippages’ of the RNA polymerase during transcription. Furin cleavage occurs intracellularly where this enzyme posttranslationally processes of several cellular hormones [20].

HPAI is of particular concern among poultry producers. While LPAI strains may cause mild illness, such as ruffled feathers, slight weight loss, and decreased egg production, HPAI can cause severe disease. HPAI is able to spread rapidly through flocks and is lethal to greater than 90% of infected poultry. This high mortality rate is attributed to the multibasic cleavage site that allows the influenza virus to spread to multiple organs within 48 h of infection [21].

Few studies have examined the prevalence of H2 influenza viruses in poultry nor have there been many studies using H2 influenza viruses for infection. However, in one study, the pathogenicity of novel H2N2 viral isolates from a duck in eastern China was used to infect poultry [22]. Ten days post-infection, none of the inoculated chickens had any clinical signs of infection. However, 9 of the 10 chickens developed serum antibodies against this LPAI H2N2 virus [22]. While only strains of H5 and H7 influenza currently contain a multicleavage site, H2 HA proteins have been shown the ability to acquire a multibasic cleavage site and become highly pathogenic in a laboratory setting [23].

Swine

In 2009, a swine-derived virus from the H1N1 subtype crossed-over to infect humans and was the cause of the 2009 pandemic. While human infections from swine influenza viruses are rare [24], sialic acid receptors for both human and avian influenza viruses are found in the swine tracheal tissues. Since pigs can be infected with both avian and human influenza viruses simultaneously, the potential for reassortment events is possible [25]. In 2006, an influenza outbreak occurred on two swine farms in Missouri. This was the first time that a novel swine H2N3 virus was identified [26]. Following isolation, characterization of this swine influenza virus identified gene segments from multiple sources, indicating that numerous reassortment events had previously occurred to generate this novel H2 virus. The HA segment was derived from a circulating H2 avian strain, while the N3 gene was most closely related to circulating avian H4N3 viruses. The remaining genes were all derived from circulating, triple reassorted swine viruses, which were originally derived from human, avian, and swine origins [26].

Previous influenza pandemics

There have been numerous influenza pandemics throughout human history. However, only the past five such pandemics in 1889, 1918, 1957, 1968, and 2009 have been characterized. The 1889 pandemics is discussed in detail below. The 1918 ‘Spanish Flu’ pandemic is the most well-known influenza pandemic. While a mystery at the time, we now know that the 1918 pandemic was caused by the H1N1 strain of influenza [27]. Overall, estimates for the number of deaths from the 1918 pandemic range from 20 to 40 million people globally [28].

The 1957 pandemic is discussed in detail below. The 1968 pandemic of ‘Hong Kong Flu’ was caused by H3N2. The number of deaths exceeded one million deaths worldwide including an estimated 100 000 deaths in the United States. The most recent Influenza pandemic occurred in 2009. This ‘Swine Flu’ pandemic was caused by a novel H1N1 virus that was created through numerous reassortment events in swine. Estimates range from 150 000 to nearly 600 000 deaths worldwide [29]. An example of a possible reassortment event between two influenza viruses is shown in Figure 2.

When two distinct influenza viruses infect the same cell, viral genome segments can combine with genome segments from the other influenza virus to produce a new influenza virus.

Figure 2.
When two distinct influenza viruses infect the same cell, viral genome segments can combine with genome segments from the other influenza virus to produce a new influenza virus.

When the red virus and the blue virus both infect the same cell, their eight respective genomic segments can be randomly packaged together instead of exclusively packaging themselves with only segments from the virus in which they were originally encapsulated. When these different segments combine, they can produce an entirely new virus as shown by the red and blue combination virus [1].

Figure 2.
When two distinct influenza viruses infect the same cell, viral genome segments can combine with genome segments from the other influenza virus to produce a new influenza virus.

When the red virus and the blue virus both infect the same cell, their eight respective genomic segments can be randomly packaged together instead of exclusively packaging themselves with only segments from the virus in which they were originally encapsulated. When these different segments combine, they can produce an entirely new virus as shown by the red and blue combination virus [1].

Pandemics

The 1889 influenza pandemic, or ‘Russian Flu’, was the first influenza pandemic with any serological data. This pandemic was characterized as a result of Louis Pasteur's and Robert Koch's work on microbes and the widely reported by newspapers that covered the pandemic [30]. While historical sources have attributed the pandemic to H2N2 [31], more recent work involving the serum samples from individuals present during the pandemic have suggested that the influenza strain was H3N8 [29]. Still other sources have stated that the 1889 pandemic was caused by H2N2 but another small pandemic occurred in 1900 and was caused by H3N8 [32]. Unfortunately, since these serum samples were obtained and stored so long ago, the identity of the 1889 pandemic influenza strain may never be definitively known. The first cases of Influenza were reported and viruses isolated in early 1889 in central Asia, Canada, and Greenland. By October, the virus had reached the urbanized areas of Moscow and St. Petersburg [30]. By some estimates, nearly 150 000 people in Constantinople and nearly half the population of St. Petersburg had contracted the virus by the year's end [30]. From Russia, the virus spread quickly across Europe [30]. The rapid spread was most likely due to a combination of factors including the massive increase in the population of cities and the development of railways over the course of the 1800s allowing for influenza viruses to spread to distant and heavily populated locations in a short period of time [30]. Over one million influenza virus associated deaths occurred between 1889 and 1891 [30].

H2N2 influenza virus pandemic of 1957

In 1957, a new viral subtype emerged and began circulating in the human population. This new influenza subtype completely replaced all of the circulating H1N1 strains that had been circulating in humans since 1918 [33]. This replacement resulted in the second pandemic of the twentieth century and was termed ‘The Asian Flu’. These new H2N2 viruses were the result of either a single reassortment or multiple reassortment events between avian H2N2 viruses containing HA, NA, and PB1 and the circulating human H1N1 viruses [34]. Since few people in 1957 had pre-existing immunity to the H2N2 viruses, these influenza viruses spread rapidly in the human population. These new viruses first infected people in East Asia and rapidly spread to people around the world resulting in ∼2 million deaths worldwide [35]. Early identification of the virus led to >60 million doses of vaccine rapidly being produced in the U.S.A. within a few months of the initial outbreak [36]. In 1968, H2 influenza viruses reassorted with H3 influenza viruses to create a new human influenza virus H3N2. This H3N2 replaced H2N2 viruses in the human population, but H2 viruses still circulate in avian species. The overwhelming majority of the currently circulating H2 influenza viruses in avian species are either H2N3 or H2N2 subtype viruses, although H2N1, H2N5, H2N7, and H2N9 are also currently in circulation. The vast majority of these are circulating in waterfowl and shorebirds, such as mallards and ducks [17].

H2 HA structure, sialic acid binding and antigenic sites

Comparisons of avian and human H2 HA crystal structures revealed a ∼90% amino acid similarity between the two structures. The superimposed images of a human and an avian H2 influenza HA proteins indicate conserved binding residues are at positions 98, 136, 153, and 183 using the H3 numbering system (Figure 3). Amino acids involved with receptor binding specificity are located at positions 186, 190, 194, 226, and 228 [37]. The H2 influenza HA structure is similar to the H1 HA protein, while the binding and antigenic profile of H2 HA molecule is similar to the H3 HA protein. The H2 and H3 HA proteins contain the same glutamine to leucine mutation at position 226 and glycine to serine amino acid at position 228 that change their preferential binding from the avian 2,3 sialic acid to the human 2,6 sialic acid binding pattern [37]. The H1 HA protein does not contain these avian to human mutations [38].

Sialic acid binding of H2 HA proteins from human and avian isolates.

Figure 3.
Sialic acid binding of H2 HA proteins from human and avian isolates.

(A) Interactions of an avian H2 HA (upper panels) and a human H2 HA (lower panels) with avian and human receptor analogues. The three secondary structure elements of the binding site, the 130- and 220-loops and the 190-helix are labeled in this backbone representation together with some selected side chains in stick representation. The broken lines indicate potential hydrogen bond interaction. In all four panels, the sialosaccharides are colored yellow for carbon atoms, blue for nitrogen, and red for oxygen, water molecules are indicated by red spheres. A/dk/Ontario/77 H2 HA, colored blue, in complex with the avian receptor, LSTa, (A) and human receptor, LSTc. (B). A/Singapore/1/57 H2 HA, colored in green, in complex with the human receptor (C) and avian receptor (D). The black arrows in (AC) indicate that for the two human receptor complexes the Sia-1/Gal-2 linkage adopts a cis conformation, whereas for the avian complex it adopts a trans conformation. Reprinted with permission. [37].

Figure 3.
Sialic acid binding of H2 HA proteins from human and avian isolates.

(A) Interactions of an avian H2 HA (upper panels) and a human H2 HA (lower panels) with avian and human receptor analogues. The three secondary structure elements of the binding site, the 130- and 220-loops and the 190-helix are labeled in this backbone representation together with some selected side chains in stick representation. The broken lines indicate potential hydrogen bond interaction. In all four panels, the sialosaccharides are colored yellow for carbon atoms, blue for nitrogen, and red for oxygen, water molecules are indicated by red spheres. A/dk/Ontario/77 H2 HA, colored blue, in complex with the avian receptor, LSTa, (A) and human receptor, LSTc. (B). A/Singapore/1/57 H2 HA, colored in green, in complex with the human receptor (C) and avian receptor (D). The black arrows in (AC) indicate that for the two human receptor complexes the Sia-1/Gal-2 linkage adopts a cis conformation, whereas for the avian complex it adopts a trans conformation. Reprinted with permission. [37].

When the HA molecule from avian H2 influenza strains binds to 2,3 sialic acids, the strongest interaction occurs between the glutamine at position 226 on HA and the Sia-1 and Gal-2 rings on the sialic acid (Figure 3A). In addition, the pentasaccharide on the sialic acid adopts a trans conformation around its glycosidic bond. This conformation allows the 2,3 sialic acid to fit snuggly into the avian H2 HA binding pocket. In contrast, when the avian H2 HA interacts with the human 2,6 sialic acid receptor, the conformation of the sialic acid saccharide shifts to a cis conformation around its glycosidic bond. This cis conformation prevents the pentasaccharide of the sialic acid from fully entering the binding pocket. This interaction also depends on more water molecules to facilitate the hydrogen bond interactions between the 2,6 sialic acid and the binding pocket when compared with the avian sialic acid [37] (Figure 3B).

When human H2N2 influenza strains bind to 2,6 sialic acids, the strongest interactions occur with the leucine residue at position 226. The leucine creates a hydrophobic area that creates an unfavorable environment for binding with the 2,3 sialic acid. This unfavorable environment affects the binding of the HA proteins from H2N2 viruses isolated in humans by creating a weaker interaction to the 2,3 sialic acid than the 2,6 sialic acid molecules [37]. These leucine interactions cause the glycosidic bond on the sialic acid to adopt a cis conformation that allows binding between the HA and the sialic acid molecule without the presence of water (Figure 3C,D). This binding pattern and ligand conformation are similar to the binding pattern of H3 hemagglutinin proteins [39]. The antigenic sites on the HA from H2 isolates resemble the more distantly related H3 HA, despite the H2 influenza HA proteins close relatedness to the HA from H1N1 isolates. This is most likely one of the major factors in antigenic sites and receptor binding sites in the H2 HA protein resembling H3 HA instead of H1 HA (Table 1) [37,40].

Table 1
Hemagglutinin from the H2 subtype have six identified antigenic sites using monoclonal antibody epitope mapping [37,40]
H2 HA antigenic sites
Site ASite ASite BSite BSite CSite CSite DSite DSite ESite ESite F
1151 132 123 181 34 274 88 207 48 83 15 
117 133 124 182 35 275 89 208 50 84 19 
118 135 148 183 36 276 91 209 53 85 30 
119 136 150 184 37 285 94 210 54 86 31 
120 137 151 185 38 290 95 211 58 87  
121 138 152 187 40 293 96 212 66 88  
125 139 153 188 41 295 110 213 69 91  
126 140 154 189 42 296 114 214 71 94  
127 141 155 191 43 300 162 217 72 102  
128 145 157 192 44 301 165 219 73 255  
129 147 158 193 45 303 166 221 74 256  
130 163 160 195 46 304 167 222 75 257  
131    269 305 168 223 78 258  
    271 306 169 224 79 259  
    272 307 170 225 80 261  
    273 308 171 233   
      172 234   
      174 235   
      177 237   
      195 239   
      196 241   
      198 242   
      202 243   
      203 245   
      204 247   
H2 HA antigenic sites
Site ASite ASite BSite BSite CSite CSite DSite DSite ESite ESite F
1151 132 123 181 34 274 88 207 48 83 15 
117 133 124 182 35 275 89 208 50 84 19 
118 135 148 183 36 276 91 209 53 85 30 
119 136 150 184 37 285 94 210 54 86 31 
120 137 151 185 38 290 95 211 58 87  
121 138 152 187 40 293 96 212 66 88  
125 139 153 188 41 295 110 213 69 91  
126 140 154 189 42 296 114 214 71 94  
127 141 155 191 43 300 162 217 72 102  
128 145 157 192 44 301 165 219 73 255  
129 147 158 193 45 303 166 221 74 256  
130 163 160 195 46 304 167 222 75 257  
131    269 305 168 223 78 258  
    271 306 169 224 79 259  
    272 307 170 225 80 261  
    273 308 171 233   
      172 234   
      174 235   
      177 237   
      195 239   
      196 241   
      198 242   
      202 243   
      203 245   
      204 247   

Site A = bold; Site B = italic; Site C = underline; Site D = bolditalic; Site E = boldunderline; Site F = italicunderline.

1

Specific amino acids in the HA sequence associated with each antigenic site are listed in underneath each specific antigenic domain. H3 HA numbering was used for the amino acid numbering.

N2 from 1957 to present

Neuraminidase is essential to the influenza virus infection cycle. Without NA, the influenza virus is able to infect and replicate in a host cell, but it is not able to spread to neighboring cells because the newly formed virions cannot escape from the originally infected cell [40]. N2 entered the human population in 1957 with the H2N2 pandemic. In 1957, H2N2 viruses preferentially bound to 2,3 sialic acid [41]. Despite the N2 NA re-pairing with an H3 HA during the 1968–1969 influenza season, these H3N2 viruses did not evolve to preferentially bind to the human 2,6 sialic acid until 1975 [42]. The antigenic sites in NA were mapped using a combination of X-ray crystallography and monoclonal antibody analysis [4345]. The NA molecule has three antigenic sites that surround the binding pocket [46]. The amino acids for each of these antigenic sites are outlined in Table 2 [43,45].

Table 2
The three NA antigenic sites of N2 mapped by monoclonal antibodies
N2 antigenic sites
Site A*Site A*Site BSite CSite C
3831 392 197 328 344 
384 393 198 329 346 
385 394 199 330 347 
386 399 200 331 357 
387 400 221 332 358 
389 401 222 334 359 
390 403  336 366 
391   338 367 
   339 368 
   341 369 
   342 370 
   343  
N2 antigenic sites
Site A*Site A*Site BSite CSite C
3831 392 197 328 344 
384 393 198 329 346 
385 394 199 330 347 
386 399 200 331 357 
387 400 221 332 358 
389 401 222 334 359 
390 403  336 366 
391   338 367 
   339 368 
   341 369 
   342 370 
   343  

Site A = bold; Site B = italic; Site C = underline.

1Specific amino acids in the NA sequence associated with each antigenic site are listed underneath each specific antigenic site [4345].

In addition to the catalytic site on NA, avian NA proteins contain an additional site that can bind to sialic acids called the hemadsorption site. This hemadsorption site is not generally observed in neuraminidases from human influenza viruses. Even many of the H2N2 pandemic viruses that were circulating in 1957 and 1968 had mutations in the hemadsorption site that resulted in a loss of enzymatic activity. The H1N1 influenza viruses circulating during the 1918 pandemic also had no enzymatic activity in the hemadsorption site. It is unclear why influenza viruses need to lose this enzymatic activity in this site, but mutations to restore enzymatic activity in the 1918 influenza virus resulted in decreased virulence [47]. Therefore, if an avian neuraminidase were to enter the human population, it would likely need to lose this enzymatic activity before it could spread effectively in humans.

If H2N2 were to reemerge in the population, there could be some protection from pre-existing N2 antibodies from a previous H2N2 or H3N2 infection. If a laboratory outbreak were to occur today, there would likely be little N2 antibody cross-reactivity in the younger human population due to N2 antigenic drift [40,48]. In regard to an avian reassortment event, Lipkind et al. [49] demonstrated that some avian viruses containing the N2 neuraminidase showed antigenic diversity to both 1957 human H2N2 neuraminidases and 1968 H3N2 neuraminidases. However, other N2 containing avian viruses showed antigenic similarity to the N2 neuraminidases from 1957. This diversity in avian N2 neuraminidase indicates that there could be a reassortment event with an antigenically distinct avian N2 neuraminidase that would be different from any human N2 neuraminidase. This would likely lead to increased morbidity and mortality compared with a reassortment that retained the N2 from a recent human H3N2 virus due to a lack of pre-existing protective N2 antibodies in the human population.

H2N2 influenza virus biosafety reclassification

In 1968, H2N2 influenza viruses, like most circulating influenza viruses, were classified as biosafety level 2 (BSL2) pathogens based upon the majority of the human population having pre-existing immunity to H2N2 influenza isolates [50]. In 2004, the College of American Pathologists (CAP) distributed kits to ∼3700 laboratories in several countries for proficiency testing of several unknown, but relatively safe pathogens [51,52]. The kits included assays for detection of influenza viruses including human H2N2 influenza viruses collected in 1957. In March 2005, Canada's National Microbiology Laboratory in Winnipeg identified the virus from the CAP kit as a 1957 H2N2 influenza virus. The Winnipeg lab immediately notified the U.S. Center of Disease Control and Prevention (CDC), as well as the World Health Organization (WHO) [51,52], which in turn immediately notified all the laboratories where the kits were distributed and ordered each laboratory to destroy the samples containing the human isolates of H2N2 influenza virus in an effort to avert a potential laboratory outbreak.

Waning immunity in humans

At the time of this potential H2N2 outbreak, the CDC was in the process of reclassifying wild-type H2N2 viruses as BSL3 pathogens. However, after this incident, the CDC accelerated their efforts to reclassify wild-type human-associated H2N2 viruses as BSL3 pathogens [53]. The higher biosafety classification was initiated in response to the lack of pre-existing immunity to human transmissible H2N2 isolates in people under 40 years of age (Figure 4). Sera from people born after 1968 have little or no hemagglutination-inhibition (HAI) activity, whereas those individuals born before 1968 showed varying antibody titers to human transmissible H2N2 influenza viruses. The oldest people in the study had the highest average HAI titers. Today, since no individual under the age of 50 has any of pre-existing immunity to these human transmissible H2N2 isolates, there is a potential for a new pandemic caused by H2N2 influenza viruses transmitting between people in the future [54]. While a pandemic caused by an H2N2 virus from a laboratory outbreak strain would likely be less severe in older adults due to previous N2 immunity, H2 could reaasort with another NA which would leave the entire human population with no antibody protection. Given the fact that H2 influenza viruses have circulated among humans at least twice in the past, there is little doubt that H2 influenza viruses will circulate in humans again in the future. While a laboratory outbreak is possible, a more likely scenario for H2 infecting humans would be from a reassortment event where and H2 influenza virus exchanges its genome segments with another influenza virus that allows H2 to efficiently infect and spread among humans once again.

HAI activity of patients against Japan/305/1957 virus.

Figure 4.
HAI activity of patients against Japan/305/1957 virus.

PBMCs were collected from healthy individuals who had not been vaccinated form influenza in the previous 6 months. These individuals all resided in Athens, Georgia, USA (IRB#: MOD6246) HAI activity is organized by birth year. A gross Mean Titer of 5.2 correlates with a 50% reduction of infection of seasonal influenza in humans.

Figure 4.
HAI activity of patients against Japan/305/1957 virus.

PBMCs were collected from healthy individuals who had not been vaccinated form influenza in the previous 6 months. These individuals all resided in Athens, Georgia, USA (IRB#: MOD6246) HAI activity is organized by birth year. A gross Mean Titer of 5.2 correlates with a 50% reduction of infection of seasonal influenza in humans.

Current H2 vaccines

From 1957 to 1968, there were three different H2N2 influenza strains that were used in formulating live-attenuated vaccines [55]. These strains are as follows: Japan/305/1957, Japan/170/1962, and Taiwan/1/1964. There have been several recent studies detailing vaccines against H2 influenza viruses in both clinical and pre-clinical studies. In pre-clinical trials, both live-attenuated and inactivated H2 vaccines have been tested. A recent report investigated the use of a trivalent vaccine using human, avian, and swine H2 viruses. These strains included Singapore/1/1957, A/Duck/HongKong/319/1978, and A/Swine/Missouri/2124514/2006 [56]. Another recent report investigated the use of A/duck/Hokkaido/162/2013 as an inactivated vaccine. The Hokkaido strain was used to vaccinate mice and shown to effectively protect against infection against multiple H2 viruses in vivo [57]. Additionally, the use of cold-adapted H2N2 viruses has been used as both vaccines and reassortment backbones in several live-attenuated influenza vaccine studies for decades [5860]. These cold-adapted H2N2 virus strains are Ann_Arbor/6/1960 and Leningrad/134/1957. There is also currently one NIH grant approved to investigate an H2N2 influenza vaccine in Phase 1 clinical trials. However, a recent Phase 1 clinical trial using a live-attenuated H2N2 virus California/1/1966 showed moderate effectiveness in generating H2 specific antibody titers [61].

Vaccine stockpiles and generating a broadly protective H2N2 influenza vaccine

The WHO is the global leader in resource management for responding to a pandemic. The 2009 influenza virus pandemic was no exception. While the WHO was able to mount and co-ordinate a global response to counter the 2009 H1N1 virus, there were several glaring flaws in their response. The WHO overwhelmed smaller countries with their weekly requests for large amounts of data. The organization was also criticized for ending their routine press conferences, which could have been used to disseminate important information in a more timely manner. There were also many flaws in the WHO vaccine distribution process. The most egregious shortcoming was the inability to distribute enough vaccines in a timely manner [62]. One effective way to mitigate the vaccine distribution issue is the stockpile vaccines for future pandemics.

In the U.S.A., influenza pandemic vaccine stockpiles are supervised by the Department of Health and Human Services (HHS) and the Biomedical Advanced Research and Development Authority (BARDA), which was established by the Pandemic and All-Hazards Preparedness Act in 2006 [63]. The purpose of this legislation was to support the research and development of countermeasures to specific threats, such as pandemic influenza outbreaks, including the development of vaccines and therapeutics [63]. Under BARDA's pandemic preparedness plan, the first goal was to develop vaccine production infrastructure to ensure that a vaccine against any infectious agent could be produced in large enough volumes to combat a pandemic [63]. For influenza viruses, 300 million doses could be produced within months of an outbreak or enough vaccine doses for the entire population of the U.S.A. within 6 months of an outbreak. The second goal is to generate a stockpile of ∼20 million doses of pandemic influenza vaccines [63]. Pandemic influenza vaccines for H5N1 highly pathogenic avian influenza viruses were commissioned for the establishment of a physical vaccine stockpile in 2009. These vaccines elicit antibodies to neutralize H5N1 viruses from several different distinct clades, with the potential to also protect against H5N2 and H5N6 influenza viruses [64].

The establishment of a pre-pandemic H2 influenza vaccine stockpile presents many of the same problems as establishing the H5 Influenza vaccine stockpile. Since H2N2 isolates are not currently circulating in people, stockpiled vaccines would need to elicit broadly reactive immune responses against multiple strains with an H2 hemagglutinin to protect against infection and transmission. Some broadly reactive vaccines are currently under development for multiple influenza subtypes and would be an ideal candidate for an H2 stockpiled vaccine [65] (Table 3).

Table 3
List of universal influenza vaccines in various stages of development that are discussed in this review. Each vaccine type is listed with its mechanism of action [66]
VaccineMechanism
Chimeric HA fusion inhibition, broadly reactive antibodies 
NP, M1, HA peptides B and T-cell responses 
COBRA Broadly reactive antibodies 
VaccineMechanism
Chimeric HA fusion inhibition, broadly reactive antibodies 
NP, M1, HA peptides B and T-cell responses 
COBRA Broadly reactive antibodies 

These broadly reactive vaccine strategies include chimeric HA vaccines, peptide vaccines, and computationally optimized broadly reactive antigen (COBRA) vaccines. Chimeric HA vaccines are produced by swapping stem regions of the HA molecule with globular head domains from other influenza virus subtypes [67]. For example, the globular head region from an H6 influenza virus could be paired with the stem region from an H1 virus with the goal of producing antibodies to the stem region of the HA molecule. The stem region appears to be more conserved than the globular head region, which may elicit broader reactive coverage across the influenza virus subtypes [67].

Peptide-based vaccines are another approach to broadly reactive influenza vaccines by exploiting B and T-cell epitopes on the HA protein. These epitopes are capable of stimulating not only antibody production, but also T-helper cells and cytotoxic T-lymphocytes. Activating these three different immune arms generates a more robust immune response that increases protection against future influenza virus infections [68].

COBRA HA sequences are generated using sequential layered-consensus methodology [66]. Using a set of HA sequences in a traditional single layer consensus design usually results in a sequence that closely resembles the most dominate sequence(s) within the set. The consensus sequence often does not incorporate sequences or epitopes in the HA molecule that are critical for antibody binding to HA proteins that occur less frequently in nature. However, these less frequent epitopes could be incorporated into a version of H2 influenza that develops into a human transmissible virus.

With a layered-consensus approach, the final COBRA HA sequence would not be biased toward the most common epitopes in the HA molecule, but rather have collected all the dominant epitopes for all the variants within the set of initial sequences (Figure 5). The COBRA methodology likely retains the key amino acid epitopes across all clade and sub-clades in each HA subtype. Indeed, COBRA designed methods for HA-based vaccines have effectively enhanced the breadth of protective immune responses against H5N1, H1N1, and H3N2 influenza viruses [6973]. A broadly protective COBRA vaccine for H2 influenza viruses may also elicit broadly reactive antibody responses to protect against historical laboratory strains, currently circulating strains of avian H2 influenza viruses, as well as future H2 influenza viruses that may evolve into a human transmissible strain and result in a new pandemic.

Amino acid alignment of wild-type sequences to produce a primary layer sequence.

Figure 5.
Amino acid alignment of wild-type sequences to produce a primary layer sequence.

The five representative amino acid sequences are aligned and the most common amino acid at each point is retained to create a consensus amino acid sequence shown in the bottom sequence [69].

Figure 5.
Amino acid alignment of wild-type sequences to produce a primary layer sequence.

The five representative amino acid sequences are aligned and the most common amino acid at each point is retained to create a consensus amino acid sequence shown in the bottom sequence [69].

Conclusion

H2 influenza viruses were responsible for millions of deaths and cost the global economy billions of dollars between 1957 and 1968. While viruses of the H2 subtype are not currently circulating in people, its previous stint as a seasonal influenza strain indicates that H2 influenza viruses can effectively spread among humans. This quality separates H2 from any other influenza subtype that is not currently transmitting between people today. The percentage of young people that lack pre-existing immunity to viruses with H2 HA proteins is growing. There is a rationale for establishing an H2 influenza vaccine stockpile similar to the H5 influenza stockpile. More resources should be devoted to the surveillance of influenza in avian and swine species as well as specific studies on H2 influenza viruses and the fitness of H2 HAs compared with other HAs. In addition, a massive effort should be devoted to developing an effective, broadly reactive H2 vaccine, and the implementation of a vaccine stockpile program to prepare for the next influenza pandemic caused by an H2 influenza virus.

Abbreviations

     
  • BARDA

    Biomedical Advanced Research and Development Authority

  •  
  • BSL2

    biosafety level 2

  •  
  • CAP

    the College of American Pathologists

  •  
  • CDC

    Center of Disease Control and Prevention

  •  
  • COBRA

    computationally optimized broadly reactive antigen

  •  
  • HA

    hemagglutinin

  •  
  • HAI

    hemagglutination-inhibition

  •  
  • HHS

    Department of Health and Human Services

  •  
  • HPAI

    high pathogenicity avian influenza

  •  
  • LPAI

    low pathogenicity avian influenza

  •  
  • M1

    matrix 1

  •  
  • M2

    matrix 2

  •  
  • NA

    neuraminidase

  •  
  • NEP

    nuclear export protein

  •  
  • NP

    nucleoprotein

  •  
  • NSP1

    nonstructural protein 1

  •  
  • NSP2

    nonstructural protein 2

  •  
  • PA

    polymerase basic protein

  •  
  • PB1

    polymerase basic protein 1

  •  
  • PB1-F2

    polymerase basic protein 1—FB2

  •  
  • PB2

    polymerase basic protein 2

  •  
  • UGA

    University of Georgia

  •  
  • WHO

    World Health Organization

Author Contribution

Both authors contributed to the intellectual content of the manuscript and gave final approval of the version to be published. Z.B.R. wrote the manuscript.

Funding

This work, including the efforts of T.M.R., was funded by University of Georgia (UGA) [MRA-001]. In addition, T.M.R. is supported by the Georgia Research Alliance as an Eminent Scholar.

Acknowledgements

Selected H2N2 influenza HA particles were obtained through the Influenza Reagent Resource, Influenza Division, WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention, Atlanta, GA, U.S.A. The authors also thank the many volunteers from Athens, Georgia who participated in the seasonal influenza vaccine studies (IRB#MOD00005030).

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

References

References
1
Fields
,
B.N.
,
Knipe
,
D.M.
and
Howley
,
P.M.
(
2013
)
Fields Virology
,
Wolters Kluwer Health/Lippincott Williams & Wilkins
,
Philadelphia
2
Centers for Disease Control and Prevention
. (
2017
)
Influenza (Flu)
,
Centers for Disease Control and Prevention
,
3
Malik Peiris
,
J.S.
(
2009
)
Avian influenza viruses in humans
.
Rev. Sci. Tech.
28
,
161
173
4
Causey
,
D.
and
Edwards
,
S.V.
(
2008
)
Ecology of avian influenza virus in birds
.
J. Infect. Dis.
197
,
S29
S33
5
Stallknecht
,
D.E.
,
Shane
,
S.M.
,
Kearney
,
M.T.
and
Zwank
,
P.J.
(
1990
)
Persistence of avian influenza viruses in water
.
Avian Dis.
34
,
406
411
6
Rogers
,
S.O.
,
Starmer
,
W.T.
and
Castello
,
J.D.
(
2004
)
Recycling of pathogenic microbes through survival in ice
.
Med. Hypotheses
63
,
773
777
7
Begon
,
M.
,
Hazel
,
S.M.
,
Baxby
,
D.
,
Bown
,
K.
,
Cavanagh
,
R.
,
Chantrey
,
J.
et al (
1999
)
Transmission dynamics of a zoonotic pathogen within and between wildlife host species
.
Proc. Biol. Sci.
266
,
1939
1945
8
Fergus
,
R.
,
Fry
,
M.
,
Karesh
,
W.B.
,
Marra
,
P.P.
,
Newman
,
S.
and
Paul
,
E.
)
Migratory birds and avian flu
.
Science
312
,
845
846
9
Centers for Disease Control and Prevention
. (
2017
)
Influenza (Flu)
,
Centers for Disease Control and Prevention
,
20 April 2017
, www.cdc.gov/flu/avianflu/monitoring-bird-flu.html
10
Hoye
,
B.J.
,
Munster
,
V.J.
,
Nishiura
,
H.
,
Klaassen
,
M.
and
Fouchier
,
R.A.M.
(
2010
)
Surveillance of wild birds for avian influenza virus
.
Emerg. Infect. Dis.
16
,
1827
1834
11
Krauss
,
S.
,
Obert
,
C.
,
Franks
,
J.
,
Walker
,
D.
,
Jones
,
K.
,
Seiler
,
P.
et al (
2007
)
Influenza in migratory birds and evidence of limited intercontinental virus exchange
.
PLoS Pathog.
3
,
e167
12
Kishida
,
N.
,
Sakoda
,
Y.
,
Shiromoto
,
M.
,
Bai
,
G.R.
,
Isoda
,
N.
,
Takada
,
A.
et al (
2008
)
H2N5 influenza virus isolates from terns in Australia: genetic reassortants between those of the Eurasian and American lineages
.
Virus Genes
37
,
16
21
13
Marché
,
S.
,
Houdart
,
P.
,
van den Berg
,
T.
and
Lambrecht
,
B.
(
2015
)
Multiyear serological surveillance of notifiable influenza A viruses in Belgian poultry: a retrospective analysis
.
Avian Dis.
59
,
543
547
14
Peng
,
X.
,
Wu
,
H.
,
Jin
,
C.
,
Yao
,
H.
,
Lu
,
X.
,
Cheng
,
L.
et al (
2014
)
Sequence and phylogenetic analysis of H2N7 avian influenza viruses isolated from domestic ducks in Zhejiang Province, Eastern China, 2013
.
Virus Genes
48
,
391
396
15
Wu
,
H.
,
Peng
,
X.
,
Peng
,
X.
,
Cheng
,
L.
and
Wu
,
N.
(
2016
)
Genetic and molecular characterization of a novel reassortant H2N8 subtype avian influenza virus isolated from a domestic duck in Zhejiang Province in China
.
Virus Genes
52
,
863
866
16
Piaggio
,
A.J.
,
Shriner
,
S.A.
,
CanDalen
,
K.K.
,
Franklin
,
A.B.
,
Kolokotronis
,
T.D.
and
Kolokotronis
,
S.O.
(
2012
)
Molecular surveillance of low pathogenic avian influenza viruses in wild birds across the United States: inferences from the hemagglutinin gene
.
PLoS ONE
7
,
e50834
17
Killian
,
M.L.
,
Zhang
,
Y.
,
Panigraphy
,
B.
,
Trampel
,
D.
and
Yoon
,
K.J.
(
2011
)
Identification and characterization of H2N3 avian influenza virus from backyard poultry and comparison with novel H2N3 swine influenza virus
.
Avian Dis.
55
,
611
619
18
Swayne
,
D.E.
(
2016
)
Overview of Avian Influenza – Poultry. Merck Veterinary Manual
,
Merck & Co
19
Racaniello
,
V.
2009
.
Influenza HA cleavage is required for infectivity
.
Virol. Blog
,
Columbia University, 22 June 2009
, http://www.virology.ws/2009/06/22/influenza-ha-cleavage-is-required-for-infectivity/
20
Böttcher-Friebertshäuser
,
E.
,
Klenk
,
H.D.
and
Garten
,
W.
(
2013
)
Activation of influenza viruses by proteases from host cells and bacteria in the human airway epithelium
.
Pathog. Dis.
69
,
87
100
21
Webster
,
R.G.
,
Brown
,
L.E.
and
Laver
,
W.G.
(
1984
)
Antigenic and biological characterization of influenza virus neuraminidase (N2) with monoclonal antibodies
.
Virology
135
,
30
42
22
Ma
,
M.J.
,
Yang
,
X.X.
,
Qian
,
Y.H.
,
Zhao
,
S.Y.
,
Hua
,
S.
,
Wang
,
T.C.
et al (
2014
)
Characterization of a novel reassortant influenza A virus (H2N2) from a domestic duck in Eastern China
.
Sci. Rep.
4
,
7588
23
Veits
,
J.
,
Weber
,
S.
,
Stech
,
O.
,
Breithaupt
,
A.
,
Graber
,
M.
,
Gohrbandt
,
S.
et al (
2012
)
Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly pathogenic phenotype
.
Proc. Natl Acad. Sci. U.S.A.
109
,
2579
2584
24
Centers for Disease Control and Prevention
. (
2017
)
Influenza (Flu)
,
Centers for Disease Control and Prevention
,
21 December 2017
, www.cdc.gov/flu/swineflu/keyfacts-variant.htm
25
Ito
,
T.
,
Couceiro
,
J.N.
,
Kelm
,
S.
,
Baum
,
L.G.
,
Krauss
,
S.
,
Castrucci
,
M.R.
et al (
1998
)
Molecular basis for the generation in pigs of influenza A viruses with pandemic potential
.
J. Virol.
72
,
7367
7373
PMID:
[PubMed]
26
Ma
,
W.
,
Vincent
,
A.
,
Gramer
,
M.
,
Brockwell
,
C.
,
Lager
,
K.
,
Janke
,
B.
et al (
2007
)
Identification of H2N3 influenza A viruses from swine in the United States
.
Proc. Natl Acad. Sci. U.S.A.
104
,
20949
20954
27
Centers for Disease Control and Prevention
. (
2014
)
Reconstruction of the 1918 Influenza Pandemic Virus
,
Centers for Disease Control and Prevention
28
Centers for Disease Control and Prevention
. (
2017
)
Influenza (Flu)
,
Centers for Disease Control and Prevention
,
2 November 2017
, www.cdc.gov/flu/avianflu/monitoring-bird-flu.html
29
Dowdle
,
W.R.
(
1999
)
Influenza A virus recycling revisited
.
Bull World Health Organ.
77
,
820
828
PMID:
[PubMed]
30
Kempiska-Mirosawska
,
B.
and
Woźniak-Kosek
,
A.
(
2013
)
The influenza epidemic of 1889–90 in selected European cities – a picture based on the reports of two Pozna daily newspapers from the second half of the nineteenth century
.
Med. Sci. Monit.
19
,
1131
1141
31
Influenza (1889–1890 Flu Pandemic) Genealogy Project
.
geni_family_tree, Geni
32
Lina
,
B.
(
2008
) History of Influenza Pandemics. In
Paleomicrobiology
(
Raoult
,
D.
and
Drancourt
,
M.
, eds),
Springer
,
Berlin, Heidelberg
33
Billings
,
M.
(
2005
)
The Influenza Pandemic of 1918
,
Stanford University
,
Stanford
34
Lindstrom
,
S.E.
,
Cox
,
N.J.
and
Klimov
,
A.
(
2004
)
Genetic analysis of human H2N2 and early H3N2 influenza viruses, 1957–1972: evidence for genetic divergence and multiple reassortment events
.
Virology
328
,
101
119
35
Influenza Pandemics
. (
2017
)
History of Vaccines
,
The College of Physicians of Philadelphia
36
Pike
,
J.
1957
.
Asian Flu Pandemic
,
Global Security
37
Liu
,
J.
,
Stevens
,
D.J.
,
Haire
,
L.F.
,
Walker
,
P.A.
,
Coombs
,
P.J.
,
Russell
,
R.J.
et al (
2009
)
Structures of receptor complexes formed by hemagglutinins from the Asian influenza pandemic of 1957
.
Proc. Natl Acad. Sci. U.S.A.
106
,
17175
17180
38
Qi
,
L.
,
Kash
,
J.C.
,
Dugan
,
V.G.
,
Wang
,
R.
,
Jin
,
G.
,
Cunningham
,
R.E.
et al (
2009
)
Role of sialic acid binding specificity of the 1918 influenza virus hemagglutinin protein in virulence and pathogenesis for mice
.
J. Virol.
83
,
3754
3761
39
Eisen
,
M.B.
,
Sabesan
,
S.
,
Skehel
,
J.J.
and
Wiley
,
D.C.
(
2002
)
Binding of the influenza A virus to cell-surface receptors: structures of five hemagglutinin–sialyloligosaccharide complexes determined by X-ray crystallography
.
Virology
232
,
19
31
40
Okuno
,
Y.
,
Isegawa
,
Y.
,
Sasao
,
F.
and
Ueda
,
S.
(
1993
)
A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains
.
J. Virol
67
,
2552
2558
PMID:
[PubMed]
41
Air
,
G.M.
(
2012
)
Influenza neuraminidase
.
Influenza Other Respir. Viruses
6
,
245
256
42
Baum
,
L.G.
and
Paulson
,
J.C.
(
1991
)
The N2 neuraminidase of human influenza virus has acquired a substrate specificity complementary to the hemagglutinin receptor specificity
.
Virology
180
,
10
15
43
Webster
,
R.G.
,
Hinshaw
,
V.S.
and
Laver
,
W.G.
(
2004
)
Selection and analysis of antigenic variants of the neuraminidase of N2 influenza viruses with monoclonal antibodies
.
Virology
117
,
93
104
44
Lee
,
J.T.
and
Air
,
G.M.
(
2006
)
Interaction between a 1998 human influenza virus N2 neuraminidase and monoclonal antibody Mem5
.
Virology
345
,
424
433
45
Air
,
G.M.
,
Elis
,
M.C.
,
Brown
,
L.E.
,
Laver
,
W.G.
and
Webster
,
R.G.
(
1985
)
Location of antigenic sites on the three-dimensional structure of the influenza N2 virus neuraminidase
.
Virology
145
,
237
248
46
Gulati
,
U.
,
Hwang
,
C.C.
,
Venkatramani
,
L.
,
Gulati
,
S.
,
Stray
,
S.
,
Lee
,
J.
et al (
2002
)
Antibody epitopes on the neuraminidase of a recent H3N2 influenza virus (A/Memphis/31/98)
.
J. Virol.
76
,
12274
12280
47
Uhlendorff
,
J.
,
Matrosovich
,
T.
,
Klenk
,
H.D.
and
Matrosovich
,
M.
(
2009
)
Functional significance of the hemadsorption activity of influenza virus neuraminidase and its alteration in pandemic viruses
.
Arch. Virol.
154
,
945
957
48
Dowdle
,
W.R.
,
Laver
,
W.G.
,
Galphin
,
J.C.
and
Downie
,
J.C.
(
1976
)
Antigenic relationships among influenza virua A neuraminidase (N2) antigens by immunodiffusion and postinfection neutralization tests
.
J. Clin. Microbiol.
3
,
233
238
PMID:
[PubMed]
49
Lipkind
,
M.
and
Shihmanter
,
E.
(
2001
)
Antigenic heterogeneity of N2 neuraminidases of avian influenza viruses isolated in Israel
.
Comp. Immunol. Microbiol. Infect. Dis.
18
,
55
68
50
Lakoff
,
A.
and
Collier
,
S.J.
(
2008
)
Biosecurity Interventions: Global Health & Security in Question
,
Columbia University Press
51
MacKenzie
,
D.
(
2005
)
Pandemic-causing ‘Asian Flu’ accidentally released
.
New Scientist
52
Falco
,
M.
)
Labs scramble to purge virus
.
Cable News Netw.
53
Roos
,
R.
(
2005
)
Vendor Thought H2N2 Virus was Safe, Officials say
,
CIDRAP, University of Minnesota
54
Nabel
,
G.
,
Wei
,
C.J.
and
Ledgerwood
,
J.E.
(
2011
)
Vaccinate for the next H2N2 pandemic now
.
Nature
471
,
157
158
55
“The ‘Flu.’” Biology, by John W. Kimball, Wm. C. Brown, 1994, pp. 561–584, 11 June 2018
, http://www.biology-pages.info/I/Influenza.html
56
Lenney
,
B.J.
,
Sonnberg
,
S.
,
Danner
,
A.F.
,
Friedman
,
K.
,
Webby
,
R.J.
,
Webster
,
R.G.
et al (
2017
)
Evaluation of multivalent H2 influenza pandemic vaccines in mice
.
Vaccine
35
,
1455
1463
57
SuzukiI
,
M.
,
Okamatsu
,
M.
,
Hiono
,
T.
,
Matsuno
,
K.
and
Sakoda
,
Y.
Potency of an inactivated influenza vaccine prepared from A/duck/Hokkaido/162/2013 (H2N1) against a challenge with A/Swine/Missouri/2124514/2006 (H2N3) in mice
.
J. Vet. Med. Sci.
79
,
1815
1821
58
Chen
,
G.L.
,
Lamirande
,
E.W.
,
Cheng
,
X.
,
Torres-Velez
,
F.
,
Orandle
,
M.
,
Jin
,
H.
et al
Evaluation of three live attenuated H2 pandemic influenza vaccine candidates in mice and ferrets
.
J. Virol.
88
,
2867
2876
59
Isakova-Sivak
,
I.
,
de Jonge
,
J.
,
Smolonogina
,
T.
,
Rekstin
,
A.
,
van Amerongen
,
G.
,
van Dijken
,
H.
et al (
2014
)
Development and pre-clinical evaluation of two LAIV strains against potentially pandemic H2N2 influenza virus
.
PLoS ONE
9
,
e102339
60
Talaat
,
K.R.
,
Karron
,
R.A.
,
Liang
,
P.H.
,
McMahon
,
B.A.
,
Luke
,
C.J.
,
Thumar
,
B.
et al (
2013
)
An open-label phase I trial of a live attenuated H2N2 influenza virus vaccine in healthy adults
.
Influenza Other Respir. Viruses
7
,
66
73
61
Isakova-Sivak
,
I.
,
Stukova
,
M.
,
Erofeeva
,
M.
,
Naykhin
,
A.
,
Donina
,
S.
,
Petukhova
,
G.
et al (
2015
)
H2N2 live attenuated influenza vaccine is safe and immunogenic for healthy adult volunteers
.
Hum Vaccin. Immunother.
11
,
970
982
62
Fineberg
,
H.V.
(
2014
)
Pandemic preparedness and response–lessons from the H1N1 influenza of 2009
.
N. Engl. J. Med.
370
,
1335
1342
63
Medical Countermeasures
. (
2018
)
MedicalCountermeasures.gov – PANDEMIC INFLUENZA
,
U.S. Department of Health and Human Services
64
Influenza A.
(
2013
)
(H5N1) Vaccine Stockpile and Inter-Pandemic Vaccine use
,
WHO
65
Sautto
,
G.A.
,
Kirchenbaum
,
G.A.
and
Ross
,
T.M.
(
2018
)
Towards a universal influenza vaccine: different approaches for one goal
.
Virol. J.
15
,
17
66
Kirchenbaum
,
G.A.
and
Ross
,
T.M.
(
2014
)
Eliciting broadly protective antibody responses against influenza
.
Curr. Opin. Immunol.
28
,
71
76
67
Krammer
,
F.
,
Pica
,
N.
,
Margine
,
I.
and
Palese
,
P.
(
2013
)
Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies
.
J. Virol.
87
,
6542
6550
68
Adar
,
Y.
,
Singer
,
Y.
,
Levi
,
R.
,
Tzehoval
,
E.
,
Perk
,
S.
,
Banet-Noach
,
C.
et al (
2009
)
A universal epitope-based influenza vaccine and its efficacy against H5N1
.
Vaccine
27
,
2099
2107
69
Giles
,
B.M.
,
Bissel
,
S.J.
,
DeAlmeida
,
D.R.
,
Wiley
,
C.A.
and
Ross
,
T.M.
(
2012
)
Antibody breadth and protective efficacy are increased by vaccination with computationally optimized hemagglutinin but not with polyvalent hemagglutinin-based H5N1 virus-like particle vaccines
.
Clin. Vaccine Immunol.
19
,
128
139
70
Giles
,
B.M.
,
Crevar
,
C.J.
,
Carter
,
D.M.
,
Bissel
,
S.J.
,
Schultz-Cherry
,
S.
,
Wiley
,
C.A.
et al (
2012
)
A computationally optimized hemagglutinin virus-like particle vaccine elicits broadly reactive antibodies that protect nonhuman primates from H5N1 infection
.
J. Infect. Dis.
205
,
1562
1570
71
Giles
,
B.M.
and
Ross
,
T.M.
(
2011
)
A computationally optimized broadly reactive antigen (COBRA) based H5N1 VLP vaccine elicits broadly reactive antibodies in mice and ferrets
.
Vaccine
29
,
3043
3054
72
Carter
,
D.M.
,
Darby
,
C.A.
,
Lefoley
,
B.C.
,
Crevar
,
C.J.
,
Alefantis
,
T.
,
Oomen
,
R.
et al (
2016
)
Design and characterization of a computationally optimized broadly reactive hemagglutinin vaccine for H1N1 influenza viruses
.
J. Virol.
90
,
4720
4734
73
Allen
,
J.D.
and
Ross
,
T.M.
(
2018
)
H3n2 influenza viruses in humans: viral mechanisms, evolution, and evaluation
.
Hum. Vaccin. Immunother.
14
,
1840
1847