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How does the new H1N1 swine virus infect humans compared to the common influenza virus?
Pandemic influenza viruses cause significant mortality in humans. In the 20th century, there are 3 influenza viruses which caused major pandemics: the 1918 H1N1 virus, the 1957 H2N2 virus, and the 1968 H3N2 virus. All three aforementioned pandemics were caused by viruses containing human adapted PB2 genes. In March and early April 2009, a new swine-origin influenza A (H1N1) virus (S-OIV) emerged in Mexico and the United States. During the first few weeks of strain surveillance, the virus spread worldwide to many countries by human-to-human transmission (and perhaps due to the airline travel). In 2 months' time, 33 countries had officially reported 5.728 cases resulting in 61 deaths, and by June 2009 WHO reported 30 000 confirmed cases in 74 countries. On June 11 of 2009, this led the World Health Organization (WHO) to raise its pandemic alert to level 5 (Human-to-human spread of the virus into at least 2 countries in 1 WHO region) of 6 (Human-to-human spread of the virus into at least 1 other country in a different WHO region in addition to phase 5 criteria). According to the sayings of Smith et al. (2009), this virus had the potential to develop into the first influenza pandemic of the twenty-first century. In the early summer of 2009, the causes of the human infection and influenza spread among humans had still remained unknown although many publications of that period tried to elucidate this influenza outburst. For example, according to the sayings of Palese, the new H1N1 could also die out entirely. “There's a 50-50 chance it will continue to circulate”, he predicts. Conclusively, in that early period, the fuzziness of the data about this new virus's behaviour led scientists only to speculate using past data. Today the 2009 H1N1 virus has ultimately created the first influenza pandemic, has disproportionately affected the younger populations (which perhaps reflects the protection in the elderly due to their exposure to H1N1 strains before 1957), but turned out to be not highly pathogenic because the majority of cases of 2009 influenza A H1N1 are mild. Genomic analysis of the 2009 influenza A (H1N1) virus in humans indicates that it is closely related to common reassortant swine influenza A viruses isolated in North America, Europe, and Asia. Therefore, it contains a combination of swine, avian, and human influenza virus genes. More studies need be conducted to identify the unrecognized molecular markers for the ability of S-OIV A (2009 H1N1) to replicate and be transmitted in humans. As a result these additional studies would help us to determine the mechanism by which an animal influenza A virus crossed the species barrier to infect humans. Additionally, these molecular determinants can be used to predict viral virulence and pathogenicity for diagnosis.
1. LITERATURE REVIEW
“Swine flu” ”influenza A [Family Orthomyxoviridae (like influenza B and C viruses), Genus Influenzavirus A] is currently the greatest pandemic disease threat to humankind (Salomon and Webster, 2009). The incidence and spread in humans of the “swine flu” influenza A virus has raised global concerns regarding its virulence and initially regarding its pandemic potential. The main cause of the “swine flu” has been identified to be the human infection by influenza A viruses of a new H1N1 (hemagglutinin 1, neuraminidase 1) subtype, or “2009 H1N1 strain” (Soundararajan et al., 2009) that contains genes closely related to swine influenza (SI) [also called swine flu, hog flu and pig flu]. Thus, the strains of virus that cause the annual seasonal flu are different than the new swine flu viruses that emerged in the spring of 2009. Consequently, as it will be analyzed in the subsequent chapters, the new swine flu virus has a unique combination of gene segments from many different sources (a combination that has not been previously reported among swine or human influenza viruses) and specifically is thought to be a mutation of four known strains of the influenza A virus, subtype H1N1:
1. one endemic in (normally infecting) humans,
2. one endemic in birds,
3. and two endemic in pigs (swine).
According to Yoon and Janke (2002), the constant evolution of influenza A viruses through mutation and reassortment present a complex and dynamic picture which is to be unfolded in the remaining Literature Review section more specifically for the H1N1 2009 virus.
Influenza is historically an ancient disease of global dimension that causes annual epidemics and, at irregular intervals, pandemics. Influenza is an infection of the respiratory tract caused by the influenza virus (see § 1.3). When compared with the majority of other viral respiratory infections (such as the common cold), the infection by influenza often causes a more severe illness (Smith, 2003). Influenza-like illness (ILI) is defined by the CDC (Centers for Disease Control and Prevention) as fever (with temperature above 37,8°C) and either cough or some throat in the absence of any other known cause. According to Webster (1999), influenza is the paradigm of a viral disease in which the continued evolution of the virus is of paramount importance for annual epidemics and occasional pandemics of disease in humans which is attributed to the fact that the H1N1 virus does not fit to the strict definition of a new subtype for which most of the population has not any experience of previous infection (Sullivan et al, 2010) as it is justified later in this Literatute Review section (§ 1.8).
Influenza is transmitted by inhalation of microdroplets (because the transmission via large-particle droplets requires close contact which is attributed to the fact that these large-particle droplets cannot remain suspended in the air for a long period of time) of respiratory secretions, often expelled by coughing or sneezing, that contain the virus or from other bodily fluids (such as fomites, diarrheal stool etc.). The incubation period is between 1 to 5 days. Symptoms typically include fever, headache, malaise, myalgia, cough, nasal discharge, and sore throat. In severe cases of influenza, a secondary bacterial pneumonia can lead to the death of a patient (Suguitan and Subbarao, 2007).
Vaccination and antiviral treatment constitute the two major options for controlling influenza and are the most effective means of preventing influenza virus infection and further transmission in humans.
1.2.1. Pandemic Influenza
An influenza pandemic is a large-scale global outbreak of the disease, whereas an epidemic is considered more sporadic and localized. The aforementioned (in the Summary section) situation of pandemic influenza occurs when a previously circulated human influenza A virus [although all the three types (A, B, and C) of influenza viruses can infect humans)] acquires novel antigenic determinants from an animal-origin influenza virus and now can infect and propagate in humans in the absence of any pre-existing immunity (see § 1.7 for details). Several influenza subtypes have infected humans. Historical accounts led us to consider that an average of three influenza pandemics have occurred each century, at intervals ranging from 10 to 50 years (Garcia-Sastre, 2005). The three influenza pandemics which occurred in the previous (20th) century are:
1. The “Spanish” influenza pandemic of 1918 (H1N1 subtype),
2. The 1957 “Asian flu” (H2N2), and
3. The 1968 ‘‘Hong Kong flu'' (H3N2).
These pandemics resulted in high morbidity, death, and also considerable social and economic disruption. They provide health authorities information on which to base preparations for a future pandemic.The first influenza pandemic of the 21st century, due to a new strain of A(H1N1) virus, was declared on 11 June 2009 by the Director-General of the World Health Organization (WHO) [Collin et al., 2009] by raising the H1N1 flu virus pandemic alert level to phase 6 as it was mentioned in the Summary section.
Although influenza B viruses do not cause pandemics, during some epidemic years they have caused more significant mortality and morbidity than influenza A viruses (FLUAV) [Garcia-Sastre, 2005].
1.3. Influenza Virus
It was already mentioned that influenza viruses are divided into three types designated A, B, and C (according to the antigenic differences of their internal structural components as it is discussed below in the current chapter). Influenza types A and B are responsible for epidemics of respiratory illness that occur almost every winter and are often associated with increased rates for hospitalization and death. As it was mentioned in the previous chapter, influenza A virus has also the capability of developing into pandemic virus. Type C infection usually causes either a sporadic mild or asymptomatic respiratory illness or no symptoms at all (Smith, 2003).
In comparison to B and C influenza types which are specific to humans, type A viruses can have different hosts, both birds and different mammals (e.g. horses and pigs) including humans (Åsjöa and Kruse, 2007). Specifically, influenza B virus strains appear to infect naturally only humans and have caused epidemics every few years (Schmitt and Lamb, 2005). On the other hand, influenza A viruses are significant animal pathogens of poultry, horses and pigs, and multiple antigenically diverse strains exist in a aquatic wild bird reservoir (Garcia-Sastre, 2005). Migrating aquatic birds carry viruses between the continents and thereby play a key role in the continuing process of virus evolution (Murphy et al., 1999). Influenza C virus causes more limited outbreaks in humans and according to Schmitt and Lamb (2005) also infects pigs. Although influenza viruses belong to the best studied viruses, according to Haller et al. (2008), the molecular determinants, which govern the increased virulence of emerging virus strains in humans and which may be associated with their transmission and transmissibility, are presently not well understood.
Influenza viruses are negative-strand RNA viruses with a segmented genome (which replicates in the nucleus of the infected cell) belonging to the Orthomyxoviridae family. The morphology of the influenza virion is described in the next chapter. On the basis of antigenic differences influenza viruses are divided into influenza virus types A, B and C. Influenza A viruses are classified on the basis of the antigenic properties of their haemagglutinin (H or HA) and their neuraminidase (N or NA) structural spike-shaped surface glycoproteins (antigens): to date, 16HA (H1-H16) and 9NA (N1-N9) subtypes have been identified (Osterhaus et al., 2008) which gives a theoretical possibility of 144 serological subtypes. Subtypes of influenza A viruses are constantly undergoing small antigenic modifications (antigenic drift) [which is a serotypic change] due to the accumulation of point mutations in their genetic material. In addition, due to the segmented genome, genetic reassortment occurs periodically when HA and NA genetic material is exchanged between viruses, thereby causing major antigenic changes (antigenic shift) [Yoon and Janke, 2002], the emergence of a new subtype (Smith, 2003) and perhaps the potential for a pandemic outbreak. Both antigenic shift and drift are discussed in § 1.7.
The family Orthomyxoviridae, except the aforementioned influenza viruses A, B and C, also contains the Thogoto viruses. Thogoto viruses are transmitted by ticks and replicate in both ticks and in mammalian species and are not discussed as part of this assignment (Schmitt and Lamb, 2005).
1.4. Influenza Virus Virion
This paragraph describes the (belonging to the Orthomyxoviridae family) virus virion morphology. These virions are spherical or pleomorphic, 80-120 nm in diameter (see 1). Some of them have filamentous forms of several micrometers in length. The virion envelope is derived from cell membrane lipids, incorporating variable numbers of virus glycoproteins (1-3) and nonglycosylated proteins (1-2) [Fauquet et al., 2005].
1. (Left) Diagram of an Influenza A virus (FLUAV) virion in section. The indicated glycoproteins embedded in the lipid membrane are the trimeric hemagglutinin (HA), which predominates, and the tetrameric neuraminidase (NA). The envelope also contains a small number of M2 membrane ion channel proteins. The internal components are the M1 membrane (matrix) protein and the viral ribonucleoprotein (RNP) consisting of RNA segments, associated nucleocapsid protein (NP), and the PA, PB1 and PB2 polymerase proteins. NS2 (NEP), also a virion protein, is not shown (Fauquet et al., 2005).
(Right) Negative contrast electron micrograph of particles of FLUAV. The bar represents 100 nm (Fauquet et al., 2005).
The lipid envelope is derived from the plasma membrane of the cell in which the virus replicates and is acquired by a budding process (see § 1.5) from the cell plasma membrane as one of the last steps of virus assembly and growth (Schmitt and Lamb, 2005) which is initiated by an interaction of the viral proteins. Virion surface glycoprotein projections are 10-14 nm in length and 4-6 nm in diameter. The viral nucleocapsid (NP) is segmented, has helical symmetry, and consists of different size classes, 50-150 nm in length (Fauquet et al., 2005). The nucleocapsid segments (the number of which depends on the virus type) surround the virion envelope which has large glycoprotein peplomers (HA, NA, HE).
There are two kinds of glycoprotein peplomers: (1) homotrimers of the hemagglutinin protein (NA) and (2) homotetramers of the neuraminidase protein (NA) [see 1 and 2]. Influenza C viruses have only one type of glycoprotein peplomer, consisting of multifunctional hemagglutinin-esterase molecules (HE) [see § 1.4.1 for further details]. Genomic segments have a loop at one end and consist of a molecule of viral RNA enclosed within a capsid composed of helically arranged nucleoprotein (NP) as it is shown in 2 (Murphy et al., 1999).
2. Schematic representation of an influenza A virion showing the envelope in which three different types of transmembrane proteins are anchored: the hemagglutinin (HA) and the neuraminidase (NA) form the characteristic peplomers and the M2 protein, which is short and not visible by electron microscopy. Inside the envelope there is a layer of M1 protein that surrounds eight ribonucleoprotein (RNP) structures, each of which consists of one RNA segment covered with nucleoprotein (NP) and associated with the three polymerase (P) proteins (Murphy et al., 1999).
The aforementioned in the previous paragraph NP protein (arginine-rich protein of approximately 500 amino acids) is the major structural protein of the eight RNPs and it has been found to be associated with the viral RNA segments. Each NP molecule covers approximately 20 nucleotides of the viral RNAs. The NP mediates the transport of the incoming viral RNPs from the cytoplasm into the nucleus by interacting with the cellular karyopherin/importin transport machinery. In addition, the NP plays an important role during viral RNA synthesis, and free NP molecules are required for full-length viral RNA synthesis, but not for viral mRNA transcription (Palese and Garcia-Sastre, 1998).
1.4.1. Influenza Viral Proteins
Influenza A and B viruses possess eight single-stranded negative-sense RNA segments (see 2) that encode structural and nonstructural proteins [NS]:
1. Hemagglutinin (HA), a structural surface glycoprotein that mediates viral entry (see § 1.5 for further details) by binding (the HA1 fragment) to sialic acid residues (present on the cell surface) on host fresh target cells, is the main target of the protective humoral immunity responses in the human host (Suguitan and Subbarao, 2007). HA is primarily responsible for the host range of influenza virus and immunity response of hosts to the infection (Consortium for Influenza Study at Shanghai, 2009). After the binding, the virus is taken up into the cell by endocytosis. At this point, the virus is still separated by the endosomal membrane from the replication and translation machinery of the cell cytoplasm (Fass, 2003). HA is initially synthesized and core-glycosylated in the endoplasmic reticulum (ER) as a 75-79 kDa precursor (HA0) which assembles into noncovalently linked homo-trimers. The trimers are rapidly transported to the Golgi complex and reach the plasma membrane, where HA insertion initiates the process of assembly and maturation of the newly formed viral particles (33-35). Just prior to or coincident with insertion into the plasma membrane, each trimer subunit is proteolytically and posttranslationally cleaved into two glycoproteins (polypeptides), HA1 and HA2 ( 3), which remain linked by a disulfide bond (Rossignol et al., 2009) and associated with one another to constitute the mature HA spike (a trimer of heterodimers). In that way, the membrane fusion during infection is promoted. Cleavage activates the hemagglutinin (HA), making it ready to attach to receptors on target cells (Murphy et al., 1999). Conclusively and in addition, the HA undergoes various post-translational modifications during its transport to the plasma membrane, including trimerization, glycosylation, disulfide bond formation, palmitoylation, proteolytic cleavage and conformational changes (Palese and Garcia-Sastre, 1998). HA1 is the subunit distal from the virus envelope, whereas HA2 contains a hydrophobic region near the carboxy terminus that anchors the HA1-HA2 complex in the membrane ( 3) [Fass, 2003]. The HA complex is brought to the cell surface via the secretory pathway and incorporated into virions, along with a section of cell membrane, as the virus buds from the cell. HA1 is the subunit distal from the virus envelope, whereas HA2 contains a hydrophobic region near the carboxy terminus that anchors the HA1-HA2 complex in the membrane (see 3) [Fass, 2003].
3. Primary structure of influenza HA and spatial organization of subunits with respect to the membrane. Cleavage of the influenza HA precursor protein HA0 yields the two subunits HA1 and HA2. HA1 is white, the fusion peptide and transmembrane segments of HA2 are black, and the remainder of HA2 is cross-hatched. For clarity, a monomer of the HA1-HA2 assembly is shown. The amino and carboxy termini of HA2 are labelled ‘‘N'' and ‘‘C,'' respectively (Fass, 2003).
2. Neuraminidase (NA) is the other major surface glycoprotein, whose enzymatic function allows the release of newly formed virions, permits the spread of infectious virus from cell to cell, and keeps newly budding virions from aggregating at the host cell surface.
This catalytic function of the NA protein is the target of the anti-influenza virus drugs oseltamivir (Tamiflu) and zanamivir (Relenza7). Although these compounds do not directly prevent the infection of healthy cells, they limit the release of infectious progeny viruses thus inhibiting their spread and shortening the duration of the illness. These NA inhibitors are effective against all NA subtypes among the influenza A viruses and may be the primary antiviral drugs in the event of a future pandemic as it proved true in the current “swine flu” influenza A outbreak. Antibodies to the NA protein do not neutralize infectivity but are protective (Suguitan and Subbarao, 2007).
Influenza C viruses lack an NA protein, and all attachment, entry and receptor destroying activities are performed by the aforementioned single spike glycoprotein: hemagglutinin-esterase-fusion (HEF) protein (Garcia-Sastre, 2005). The HEF protein distinguishes the antigenic variants of the genus C of the Orthomyxoviridae family, and the antibody to HEF protein neutralizes infectivity (Schmitt and Lamb, 2005). Of the three virus types, A and B viruses are much more similar to each other in genome organization and protein homology than to C viruses, which suggests that influenza C virus diverged well before the split between A and B viruses (Webster, 1999).
Three proteins comprise the viral polymerase of the influenza viruses: two basic proteins (PB1 and PB2) and an acidic protein (PA). They are present at 30 to 60 copies per virion. The RDRP (RNA-dependent RNA polymerase) complex consists of these 3 polymerase proteins (Lamb and Krug, 2001). Together with the aforementioned scaffold protein NP (helically arranged nucleoprotein), these three polymerase proteins associate with the RNA segments to form ribonucleoprotein (RNP) complexes (Murphy et al., 1999). Thus, the RNPs contain four proteins and RNA. Each subunit of NP associates with approximately 20 bases of RNA (Lamb and Krug, 2001). The RNP strands usually exhibit loops at one end and a periodicity of alternating major and minor grooves, suggesting that the structure is formed by a strand that is folded back on itself and then coiled on itself to form a type of twin-stranded helix (Schmitt and Lamb, 2005). RDRP transcribes the genome RNA segments into messenger RNAs (mRNA). The RDRP complex carries out a complex series of reactions including cap binding, endonucleolytic cleavage, RNA synthesis, and polyadenylation.
The PA protein may be involved in viral RNA replication and, in addition, the expression of the PA protein in infected cells has been associated with proteolytic activity. The functional significance of the latter activity is not yet understood (Palese and Garcia-Sastre, 1998).
Two viral RNA segments (7 and 8) encode at least two proteins each by alternative splicing. Gene segment 7 (see 4) codes for two proteins: matrix protein M1, which is involved in maintaining the structural integrity of the virion, and M2, an integral membrane (surface) protein that acts as an ion channel and facilitates virus uncoating. It is widely believed that the M1 protein interacts with the cytoplasmic tails of the HA, NA, and M2 (or BM2) proteins and also interacts with the ribonucleoprotein (RNP) structures, thereby organizing the process of virus assembly (Schmitt and Lamb, 2005).
The drugs amantadine and rimantadine bind to the influenza A M2 protein and interfere with its ability to transport hydrogen ions into the virion, preventing virus uncoating. Amantadine is only effective against influenza A viruses (Suguitsan and Subbarao, 2007). Therefore, for the antiviral therapy, there are two classes of drugs which are currently available for the chemoprophylaxis and the treatment of influenza (Rossignol et al., 2009). These include the aforementioned NA inhibitors oseltamivir and zanamivir, which impair the efficient release of viruses from the infected host cell, and amantadine and rimantadine, which target the viral M2 protein required for virus uncoating. Passively transferred antibodies to M2 can protect animals against influenza viruses, but such M2-specific antibodies are not consistently detected in human convalescent sera (Black et al., 1993), suggesting that this type of immunity may play a minor role in the clearance of influenza virus in humans.
Gene segment 8 (see 4) is responsible for the synthesis of the nonstructural protein NS1 and nuclear export protein (NEP, formerly called NS2) [Murphy et al., 1999] which is a minor structural component of the viral core and that mediates nucleo-cytoplasmic trafficking of the viral genome (Garcia-Sastre, 2005). NEP (NS2) plays a role in the export of RNP from the nucleus to the cytoplasm. NS1 protein suppresses the antiviral mechanism in host cells upon viral infection (Chang et al., 2009) and is involved in modulating the host's interferon response (Garcia-Sastre, 2005).
Recently, an unusual 87-amino acid peptide arising from an alternative reading frame of the PB1 RNA segment has been described (Chen et al., 2001). This protein, PB1-F2, is believed to function in the induction of apoptosis as a means of down-regulating the host immune response to influenza infection. Specifically, it appears to kill host immune cells following influenza virus infection. It has been called the influenza death protein (Chen et al., 2001). PB1 segment encodes this second protein from the +1 reading frame. This protein consists of 87-90 amino acids (depending on the virus strain). This protein is absent in some animal, particularly swine, virus isolates. PB1-F2 protein is not present in all human influenza viruses. Human H1N1 viruses encode a truncated version. However, it is consistently present in viruses known to be of increased virulence in humans, including the viruses that caused the 1918, 1957, and 1968 pandemics. PB1-F2 localizes to mitochondria and treatment of cells with a synthetic PB1-F2 peptide induces apoptosis9 (Neumann et al., 2008).
4. Orthomyxovirus genome organization. The genomic organization and ORFs are shown for genes that encode multiple proteins. Segments encoding the polymerase, hemagglutinin, and nucleoprotein genes are not depicted as each encodes a single protein.
(A) Influenza A virus segment 8 showing NS1 and NS2 (NEP) mRNAs and their coding regions. NS1 and NS2 (NEP) share 10 amino-terminal residues, including the initiating methionine. The open reading frame (ORF) of NS2 (NEP) mRNA (nt 529-861) differs from that of NS1.
(B) Influenza A virus segment 7 showing M1 and M2 mRNAs and their coding regions. M1 and M2 share 9 amino-terminal residues, including the initiating methionine; however, the ORF of M2 mRNA (nt 740-1004) differs from that of M1. A peptide that could be translated from mRNA has not been found in vivo.
(C) Influenza A virus PB1 segment ORFs10. Initiation of PB1 translation is thought to be relatively inefficient based on Kozak's rule, likely allowing initiation of PB1-F2 translation by ribosomal scanning (Fauquet et al., 2005).
In the same way, the M2 protein is anchored in the viral envelope of the influenza A virus, the ion channel proteins BM2 (it is encoded by a second open reading frame10 of RNA segment 7 of influenza B virus, and its function has not been determined) and CM2 are contained in influenza B and C viruses respectively ( 5). The CM2 protein is most likely generated by cleavage of the precursor protein. The influenza B viruses encode one more transmembrane protein, or NB, of unknown function (Garcia-Sastre, 2005). The cellular receptor for the influenza C virus is known to be the 9-0-acetyl-N-acetylneuraminic acid, and its receptor-destroying enzyme is not an NA, as it was already mentioned, but a neuraminate-O-acetylesterase. Like the HA protein of A and B viruses, the HEF of influenza C viruses must be cleaved in order to exhibit membrane fusion activity (Palese and Garcia-Sastre, 1998).
1.5. Viral Entry
Influenza virus infection is spread from cell to cell and from host to host in the form of infectious particles that are assembled and released from infected cells. A series of events must occur for the production of an infectious influenza virus particle, including the organization and concentration of viral proteins at selected sites on the cell plasma membrane, recruitment of a full complement of eight RNP segments to the assembly sites, and the budding and release of particles by membrane fission (Schmitt and Lamb, 2005).
Viral entry is a multistep process that follows attachment of the virion to the cellular receptor and results in deposition of the viral genome (nucleocapsid) in the cytosol (receptor-mediated endocytosis). The entry of enveloped viruses is exemplified by the influenza virus ( 6). The sequential steps in entry include (Nathanson, 2002):
§ Attachment of the HA spike [the virus attachment protein (VAP)] to sialic acid receptors (bound to glycoproteins or glycolipids) on the cellular surface (see § 1.4.1 for further details). This step contributes to pathogenesis, transmission, and host range restriction.
§ Internalization of the virion into an endocytic vacuole.
§ Fusion of the endocytic vacuole with a lysosome, with marked lowering of the pH (see 6). In endosomes, the low pH-dependent fusion occurs between viral and cell membranes. For influenza viruses, fusion (and infectivity) depends on the cleaved virion HA (FLUAV and FLUBV: HA1, HA2; FLUCV: HEF1, HEF2) [Murphy et al, 1999]. The infectivity and fusion activity are acquired by the post-translational cleavage of the HA of the influenza viruses which is accomplished by cellular proteases. Cleavability depends, among other factors, on the number of basic amino acids at the cleavage site. It produces a hydrophobic amino terminal HA2 molecule (Fauquet et al., 2005).
6. Diagram of the stepwise entry of influenza virus at a cellular level. Key events are attachment of the virion; internalization of the virion by endocytosis; lowering the pH of the endocytic vacuole leading to drastic reconfiguration of the viral attachment protein (hemagglutinin, HA1 and HA2); insertion of a hydrophobic domain of HA2 into the vacuolar membrane; fusion of the viral and vacuolar membranes; release of the viral nucleocapsid into the cytosol (Nathanson, 2002).
§ A drastic alteration in the structure of the HA1 trimer, with reorientation of the HA2 peptide to insert its proximal hydrophobic domain into the vacuolar membrane (Nathanson, 2002).
§ Fusion of viral and vacuolar membranes (Nathanson, 2002).
§ Integral membrane proteins migrate through the Golgi apparatus to localized regions of the plasma membrane (Fauquet et al., 2005).
§ New virions form by budding, thereby incorporating matrix protein and the viral nucleocapsids which align below regions of the plasma membrane containing viral envelope proteins. Budding is from the apical surface in polarized cells (Fauquet et al., 2005).
§ Release of the viral nucleocapsid into the cytosol: After the formation of fusion pores, viral ribonucleoprotein complexes (RNPs) are delivered into the cytosol. RNPs are then transported into the nucleus, where transcription and replication occurs (see 7) [Garten and Klenk, 2008].
How the replication and the transcription of the genome of influenza virus take place in the nuclei of infected cells is summarized in detail by Palese and Garcia-Sastre (1998) [ 7].
(1) Adsorption: the virus interacts with sialic acid-containing cell receptors via its HA protein, and is intenalized by endosomes.
(2) Fusion and uncoating: the HA undergoes a conformational change mediated by the acid environment of the endosome, which leads to the fusion of viral and cellular membranes. The inside of the virus also gets acidified due to proton trafficking through the M2 Ion channel. This acidification is responsible for the separation of the M1 protein from the ribonucleoproteins (RNPs), which are then transported into the nucleus of the host cell thanks to a nuclear localization Signal in the NP.
(3) Transcription and replication: the viral RNA (vRNA) is transcribed and replicated in the nucleus by the viral polymerase. Two different species of RNA are synthesized from the vRNA template:
(a) full-length copies (cRNA), which are used by the polymerase to produce more vRNA molecules; and
(4) Translation: following export into the cytoplasm the mRNAs are translated to form viral proteins. The membrane proteins (HA, NA and M2) are transported via the rough endoplasmic reticulum (ER) and Golgi apparatus to the plasma membrane. The viral proteins possessing nuclear signals (PB1, PB2, PA, NP, M1, NS1 and NEP) are transported into the nucleus.
(5) Packaging and budding: the newly synthesized NEP protein appears to facilitate the transport of the RNPs from the nucleus into the cytoplasm by bridging the RNPs with the nuclear export machinery. M1-RNP complexes are formed which interact with viral proteins in the plasma membrane. Newly made viruses bud from the host cell membrane (Palese and Garcia-Sastre, 1998).
1.5.1. Sialic Acid Receptors of Influenza Viruses
Sialic acids (Sias) are a family of negatively charged 9-carbon sugars typically occurring at the terminal positions of glycoconjugates on the cell surface and secreted molecules of the deuterostome branch of the animal kingdom. Therefore, Sialic acid is a sugar group commonly found on a variety of glycosylated molecules. Because of the highly exposed location of sialic acids, sialic acids often serve as recognition epitopes (antigenic sites) for endogenous lectins, such as selectins and siglecs, and as components of attachment sites utilized by microbial pathogens (Matrosovich et al., 2008).
More than 40 known sialic acid (Sia) species differ from each other by substituents at the amino group (N5) and at four hydroxyl groups (O4, O7, O8, and O9). Influenza A and B viruses use as a receptor molecule the most common derivative and a biosynthetic precursor of other family members, non-Oacetylated N-acetylneuraminic acid (Neu5Ac) [Matrosovich et al., 2008].
Sialic acids are represented in vivo in the oligosaccharide chains of glycoproteins and glycolipids (gangliosides) (Matrosovich et al., 2008). The affinity of HA for sialic-acid-containing molecules on target cells strongly determines the host-range restriction among influenza A viruses (Suguitan and Subbarao, 2007). Virus binding depends not only on HA affinity for the terminal Sia residues, but also on the structure of the underlying oligosaccharide and protein or lipid moieties of the receptors (Matrosovich et al., 2008).
In natural glycoconjugates, sialic acids are
1. α2-3- or α2-6-linked to Gal and GalNAc,
α2-6-linked to GlcNAc, or
α2-8-linked to the second sialic acid (Sia) residue.
Influenza viruses generally do not bind to α2-8-linked Neu5Ac moieties and can recognize only α2-3- or α2-6-linked sialic acid (Sia) epitopes (Neu5Acα2-3/6Gal, Neu5Acα2-3/6GalNAc, and Neu5Acα2-6GlcNAc) [Matrosovich et al., 2008].
1.6. Viral Infectivity Factors
All available evidence indicates that FLUAV virulence cannot be attributed to a single factor but is in fact multifactorial. It requires an optimal combination of several genetic traits. This optimal combination is usually the product of evolutionary pressure which optimizes functional interactions between viral components and between viral and host factors. Haller et al. (2008) give as an example a number of virulence determinants that have been identified. These comprise receptor binding and cleavability of the viral hemagglutinin (HA), activity of the neuraminidase (NA), compatibility between HA and NA and the efficacy of the viral polymerase complex, among others (Haller et al., 2008). An example how the alteration of one of the factors above can affect the transmitability to humans is given by Murphy et al. (1999). It constitutes the most striking lesson of how easily hemagglutinin cleavability can be altered and virulence changed. This example comes from the analysis of an outbreak of avian influenza in Pennsylvania in 1983. Early in the year, a virus (H5N2) was introduced from wild birds into chicken flocks, producing a mortality of less than 10%. However, in October 1983, the accumulation of about seven point mutations in the hemagglutinin gene of this virus resulted in an increase in mortality to over 80% and a coincident increase in viral transmissibility - all contributing to an epidemic that cost the regional poultry industry more than 60 million dollars. One of the mutations abolished a glycosylation site on the viral hemagglutinin molecule, thus exposing the HAl/HA2 cleavage site that had previously been concealed by a carbohydrate side chain.
1.7. Antigenic Variation (Shift/Drift)
Influenza (flu) virus is infamous for its antigenic variation characterized by Fass (2003) as the superficial changes that help the virus avoid detection by the immune system while preserving its essential functions. Although antibodies neutralize flu virus, rapid variation in the sequence of the influenza surface proteins causes recurrent outbreaks and the inability to develop a permanent vaccine against the virus (Fass, 2003). As a result, vaccines must change every year. The extensive antigenic variation exhibited by the HA and NA surface proteins of human influenza viruses contributes to their evolutionary success as they undergo genetic change to elude the host's immune responses (Suguitan and Subbarao, 2007). The phenomenon of antigenic variation is observed mainly with RNA viruses which use low fidelity RNA polymerases for replication (Wagner et al., 2005). These variations are brought about by two fundamental mechanisms: antigenic drift and antigenic shift (Suguitan and Subbarao, 2007). Antigenic drift involves minor antigenic changes in the HA and NA, whereas antigenic shift involves major antigenic changes in these molecules resulting from replacement of the gene segment (Webster, 1999). Type A viruses undergo both kinds of changes, whereas influenza type B viruses change only by the more gradual process of antigenic drift (Smith, 2003).
When the two constituents of HA (HA1 and HA2) were separated and studied by peptide mapping, it was discovered that antigenic change occurred in the HA1 molecule almost entirely (Oxford et al., 2003).
To reflect the high degree of antigenic diversity among influenza A viruses, a system of designating virus isolates has been developed. The current system of nomenclature contains two parts: (1) type and strain designation and, for influenza A viruses, (2) a description of the antigenic specificity of the HA and NA antigens.
The strain designation for influenza virus type A contains information on the nucleoprotein antigenic type, the host of origin if isolated from nonhuman species (e.g. swine), geographical origin, strain number, and the year of isolation. For the influenza A viruses, the antigenic description indicating HA and NA antigens follows the strain designation in parentheses (Yoon and Janke, 2002). Thus, an example of an isolate of swine influenza is designated A/Swine/Iowa/15/30 (H1N1).
There is no provision for describing distinct subtypes of B and C viruses.
1.7.1. Antigenic Drift
Antigenic drift, which occurs within a subtype, results from the gradual accumulation of point mutations in the HA and NA proteins of influenza viruses that arise from a combination of the inherently low fidelity of the viral RNA-dependent polymerase complex (lacking proofreading ability) and from positive selection driven by the antibody response of the host (Suguitan and Subbarao, 2007). The seasonal influenza epidemics occur as a result of genetic drift (Åsjöa and Kruse, 2007).
Changes in the hemagglutinin are clustered in five regions of the molecule, which correspond to important antigenic sites. Substitution of a single amino acid in a critical antigenic site may abolish the capacity of the antibody to bind to that site (Murphy et al., 1999). The high mutation rate seems to alter the antigenicity of the virus, thus escaping B- and T-cell recognition and antiviral antibodies (Wagner et al., 2005).
Thus, antigenic drift is an ongoing process of evolution that permits epidemic influenza A and B viruses to evade neutralization by antibodies elicited by prior infection or immunization. For that reason, influenza vaccines are updated annually to keep pace with antigenic drift so that the virus included in the vaccine formulation will closely match that of the current year's epidemic circulating strain (Suguitan and Subbarao, 2007).
Antigenic drift apparently does not occur among influenza viruses C. However, antigenic variation between distinct co-circulating lineages has been detected in HI tests with both anti-HEF Mabs (Monoclonal antibodies) and polyclonal antisera (Fauquet et al., 2005). The antigenic drift can be simulated in the laboratory by the growth of the influenza viruses in the presence of monoclonal antibodies to a single antigenic site (Webster, 1999).
1.7.2. Antigenic Shift
While antigenic drift is a continuous process of change, antigenic shift arises less frequently, results in greater antigenic change, and is only met with influenza A viruses (Suguitan and Subbarao, 2007). It is defined as a sudden and dramatic change in the antigenicity of a virus ( 8) which is owing to the reassortment of the segmented virus genome with another genome of a different antigenic type (Cann, 2005). Specifically, it occurs as a result of the introduction into the human population of a novel HA and/or NA protein that is immunologically distinct from the influenza A viruses circulating in recent years (Cox and Subbarao, 1999). The segmented nature of the influenza genome permits the possible exchange of gene segments (in a process referred to as genetic reassortment) when two different influenza A viruses infect the same cell at the same time. The isolation of viruses in nature with different combinations of HA and NA suggests that such genetic reassortments occur freely and frequently (Suguitan and Subbarao, 2007). This phenomenon will not happen very often, but if it does, one result of the mixed infection will be the generation of a new hybrid virus that might have, say, a hemagglutinin membrane glycoprotein from one parent and all the other components from the human virus. To add to this, swine influenza virus strains recognize some of the human cell receptors utilized by their human influenza A virus counterparts. This means that in a farm where pigs are intensely cultivated, a multiple infection could involve a swine virus as well as a human virus.
The problem with antigenic shift is even more complicated by the fact that pigs (but not humans) have efficient receptors for avian influenza viruses. Therefore, a multiple infection in pigs with different avian strains or avian and porcine strains can lead to a very significant random reassortment of different markers. This can happen with some frequency in areas in which there is very intense farming and animal husbandry in relatively limited spaces, which is typical of many small farms in East Asia where pigs, ducks, chickens, and other animals are all tended together (Wagner et al., 2008). The movement of live pigs between Eurasia and North America seems to have facilitated the mixing of diverse swine influenza viruses, leading to the multiple reassortment events associated with the genesis of the S-OIV (Swine-origin Influenza Virus) strain. Domestic pigs have been described as a hypothetical “mixing-vessel”, mediating by the aforementioned reassortment the emergence of new influenza viruses with avian or avian-like genes into the human population, and triggering a pandemic associated with antigenic shift (Smith et al., 2009). The reason can be attributed to the fact that pigs are known to be susceptible to influenza viruses of both avian and mammalian (including human) origin because their tracheal epithelium contains virus receptors for both of them (Yoon and Janke, 2002). Specifically, this susceptibility is due to the presence of both α2,3- and α2,6-galactose sialic acid linkages in cells lining the pig trachea which can result in modification of the receptor-binding specificities of avian influenza viruses from α2,3 to α2,6 linkage, which is the native linkage in humans, thereby providing a potential link from birds to humans (Brown, 2008). Therefore, because pigs support replication of both avian and human viruses, they were considered and proved to be a plausible intermediate host for the generation of human pandemic strains by gene reassortment (Matrosovich et al., 2008).
Upon antigenic shift, the resulting successful virus is essentially a “new” virus, and is relatively unaffected by the immune defenses mounted against earlier forms of virus. Thus, the new virus can spread throughout the human population despite the high level of immunity to prior forms of influenza A (Wagner et al., 2008).
The immunological variation of various flu virus proteins from virus isolated over a considerable period of time is shown in 9.
9. Antigenic changes in the surface glycoproteins of influenza A virus between 1918 and 1980. Abrupt changes in these antigens (antigenic shifts) are the result of mixed infections and random assortment of nucleocapsids to generate novel genotypes. Such shifts, which occur with random frequency, lead to epidemics worldwide. Strain designations at the bottom of the indicate hemagglutinin (H) and neuraminidase (N) genotypes.
1.8. Origins and Historical Perspective of Swine Influenza Virus (SIV) and Swine - Origin Influenza Virus (S-OIV)
It is not generally appreciated by the international scientific community and the bibliography that descendents of the H1N1 influenza A virus that caused the catastrophic and historic pandemic of 1918-1919 have persisted in humans for more than 90 years and have continued to contribute their genes to new viruses, causing new pandemics, epidemics, and epizootics (Morens et al., 2009). In fact, the current international pandemic caused by a novel influenza A (H1N1) virus derived from two unrelated swine viruses, one of them a derivative of the 1918 human virus [A/South Carolina/1/1918 (SC18)], adds to the complexity surrounding this persistent progenitor virus, its descendants, and its several lineages (Morens et al., 2009).
Before 1918, influenza in humans was well known, but the disease had never been described in pigs (Zimmer and Burke, 2009). Swine influenza (SI) is unique in the sense that it has been both a historically significant and, as it proved by the current outbreak, an ever-present disease which constantly presents newly emerging disease problems (Olsen, 2002). Just as the 1918 pandemic spread the human influenza A virus worldwide and killed 40 million to 50 million people, herds of swine were hit with a respiratory illness that closely resembled the clinical syndrome affecting humans (Zimmer and Burke, 2009). Recent evidence indicates that the early swine and human isolates were closely related H1N1 viruses that had recently emerged from an avian source (Olsen, 2002).
The gene pool of influenza A viruses in aquatic birds (avian influenza reservoir) provides all the genetic diversity (antigenic drift and genetic shift) required for the emergence of pandemic influenza viruses for humans, lower animals and birds (Webster, 1999). Avian influenza A viruses seem to exist as transient complexes of eight genes (see 10) that assemble and reassemble promiscuously, if not randomly, in an enormous global avian reservoir (Morens et al., 2009). Within this reservoir, avian viruses remain stably adapted to the enteric tracts of hundreds of avian species (Morens et al., 2009). Moreover, the available evidence suggests that all of the pandemic influenza virus strains, including the Spanish 1918 (H1N1) [A/South Carolina/1/1918 (SC18)], Asian 1957 (H2N2) and Hong Kong 1968 (H3N2) viruses, originated from the avian influenza reservoir either by reassortment (swapping of viral genetic information in hosts coinfected with more than one influenza virus) or direct transfer. Influenza outbreaks in domestic animals, including poultry, also originate from the avian reservoir (Salomon and Webster, 2009).
A(H1N1) influenza viruses were first isolated from swine in 1930 (Garten et al., 2009). Specifically, the virus was isolated and identified in 1930 by Shope (1931). Shope also discovered that the antibody specificity against the 1918 human influenza virus rapidly appeared divergence from that of swine influenza virus (Zimmer and Burke, 2009). From 1930 to the late 1990s these “classical (North American) swine influenza” viruses circulated in swine and remained relatively antigenically stable (Garten et al., 2009).
Following the reported occurrence of influenza in pigs at the time of the 1918 pandemic, swine influenza (SI) was for a long time apparently confined to North America where it remained the predominant subtype and predominant cause of in influenza among pigs until the 1990s. These and viruses related closely are termed classical (North American) viruses (see 10 and 11) and have also been isolated widely from pigs in South America, Europe and Asia (Brown, 2008).
11. Emergence of Influenza A (H1N1) Viruses from Birds and Swine into Humans. The diagram shows the important events and processes in the emergence of influenza A (H1N1) viruses during the past 91 years. Avian, swine, and human populations are represented in the top, middle, and bottom of the diagram, respectively. Epidemic or zoonotic viruses are shown as wide horizontal arrows (white for avian viruses, light blue or pink for swine viruses, and dark blue for human viruses). Crossspecies transmissions are shown as vertical dashed lines, with thick lines for transfers that gave rise to sustained transmission in the new host and thin lines for those that were transient and resulted in a selflimited number of cases. Principal dates are shown along the bottom of the diagram. The disappearance of H1N1 in 1957 most likely represents competition by the emerging pandemic H2N2 strain in the face of population immunity to H1N1. (Zimmer and Burke, 2009).
Human influenza A (H1N1) abruptly disappeared in 1957 and was replaced by a new reassortant virus that combined genes from the H1N1 strain and an avian virus (see 10). This new influenza A (H2N2) strain contained three new segments from the avian source and maintained the other five segments from the H1N1 strain of 1918 lineage (total 8 segments). After this pandemic subtype emerged, human influenza A (H1N1) was not detected again until 1977 (Fort Dix case). Reasons for the complete disappearance of this strain in 1957 are not clear, but it is likely that high levels of existing homologous immunity, coupled with a burst of heterologous immunity from the new H2N2 strain ( 10), were sufficient to eliminate the virus (Zimmer and Burke, 2009).
In 1976 (January), swine virus infections reappeared and were observed at an army training base in Fort Dix, New Jersey (Zimmer and Burke, 2009). Scientific evidence demonstrated that the virus causing infections was similar to strains that had been circulating in pigs for several years, suggesting insufficient virulence to affect a human population of average density (Wang and Palese, 2009). The novel influenza virus that was identified was H1N1 A/New Jersey/76 was considered the cause of the epidemic that resulted in serologic evidence of 230 cases and one death. Because of careful characterization of the soldiers and the nature of basic training, the outbreak at Fort Dix provided an ideal setting for investigation and modeling of the epidemic events. The Fort Dix event had been the last major outbreak of S-OIV in humans till the recent re-emergence of S-OIV (Smith et al., 2009).
Swine influenza (SI) demonstrates two major classes in its haemagglutinin (HA) protein evolution, associated with what are termed “Classic North American Swine Flu” (see paragraph above) and “Eurasian Swine Flu” (Gatherer, 2009). In addition there are other porcine influenza haemagglutinins of more uncertain affinities, possibly resulting from independent re-assortment events with avian H1 haemagglutinins. Since 1979 (see 10 and 11), the dominant H1N1 viruses in European pigs have been the “avian-like” (Eurasian Swine Flu) H1N1 viruses that are antigenically and genetically distinguishable from the classical swine H1N1 influenza viruses, but related closely to H1N1 viruses isolated from ducks (Brown, 2008). The first detected “Eurasian Swine Flu” virus was in Belgium in 1979 (Smith et al., 2009). The NA genes from the Eurasian and North American swine influenza virus lineages are highly divergent, with more than 77 differences in amino acids between these lineages. The aforementioned amino acid divergence between haemagglutinin proteins from classic and Eurasian swine flu ( 10) strains is 20-25% (Gatherer, 2009). The lineage of the “avian-like” (Eurasian Swine Flu) H1N1 virus became established and gradually replaced classical swine H1N1 viruses, and also reassorted in pigs with human H3N2 viruses (A/Port Chalmers/1/1973-like) [Smith et al., 2009]. It is worthwhile mentioning that, until now (Smith et al., 2009), there has been no evidence of Eurasian avian-like swine H1N1 circulating in North American pigs.
Finally, the unique “triple reassortant” H3N2 (or rH3N2) viruses have been isolated frequently from pigs throughout the USA since 1998 (Brown, 2008) and human infections with swine reassortant viruses have been documented (Newman et al., 2008) [12 cases of human infection with such viruses were identified in the United States from 2005 through 2009 (Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, 2009)]. The establishment of H3N2 triple reassortant virus brought the genotypic variability in classical H1N1 viruses (Brown, 2008). These viruses contain and PB1 polymerase genes of human influenza virus origin, NA (N2), HA (H1), NP, M, and NS genes of classical swine H1N1 virus origin, and PB2 and PA polymerase genes of North American avian virus origin (see 10) [Brown, 2008]. These viruses have spread to many countries and have continued to evolve with further reassortment with prevailing human H3N2 viruses. Specifically, shortly after the initial detection of the rH3N2 virus, subsequent reassortment between the rH3N2 virus and classical H1N1 swine virus is believed to have resulted in the generation of further triple reassortant swine A(H1N1) and A(H1N2) viruses. In addition to the detection of these triple reassortants in North American swine populations since 1998, triple reassortant swine viruses of the North American lineage have also recently been detected in Asian swine populations (Garten et al., 2009). Given the history of reassortment events of swine influenza, it is likely that many additional reassortant viruses have emerged but have not been sampled (Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, 2009). The emergence of the H3N2 viruses has posed significant challenges for the control of swine influenza (SI), including the need to develop new diagnostic reagents and assays, and new vaccines (Brown, 2008).
In April 2009, near the end of the usual (seasonal) influenza season in the Northern Hemisphere, the first two cases of S-OIV (Swine-origin Influenza Virus) were identified in the United States (Zimmer and Burke, 2009). These cases were confirmed to have been caused by a genetically similar swine virus that had not been previously identified in the United States. Genetic analysis of the strains showed that they were derived from a new reassortment of six gene segments from the known triple reassortant swine virus, and two gene segments (NA and matrix protein) from the Eurasian influenza A (H1N1) swine virus lineage (see 10).
1.9. 2009 Swine - Origin Influenza Virus (S-OIV) Genomics
Clinical samples that were obtained from the first 642 patients (Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, 2009) with confirmed infection (from April 15 through May 5, 2009 in United States) and that were received by the CDC (Centers for Disease Control and Prevention) were tested with the use of real-time RT-PCR assays for swine influenza, and all the samples were confirmed to be positive for S-OIV. Among the 49 S-OIV isolates from 13 states in the United States that were sequenced at the CDC as of May 5, 2009, all were 99 to 100% identical in all genes (at the amino acid level). Phylogenetic analysis of sequences of all genes of A/California/04/2009 (or Cal0409), the virus isolated from a pediatric patient with uncomplicated, upper respiratory tract illness, showed that its genome contained six gene segments (PB2, PB1, PA, HA, NP, and NS) that were similar to ones previously found in triple-reassortant swine influenza viruses circulating in pigs in North America (see § 1.8). The genes encoding neuraminidase (NA) and M protein (M) were most closely related to those in influenza A viruses circulating in swine populations in Eurasia (see 12). The largest proportion of genes in this novel virus comes from swine influenza viruses (30.6 % from North American swine influenza strains, 17.5 % from Eurasian swine influenza strains), followed by North American avian influenza strains (34.4 %) and human influenza strains (17.5 %) [Garten et al., 2009]. This particular genetic combination of influenza virus segments had not been encountered before in the United States or elsewhere. As it was mentioned in § 1.8, the North American triple-reassortant swine influenza A (H1) viruses were known to be composed of the hemagglutinin (HA), nucleoprotein (NP), NA, M, and nonstructural protein (NS) genes, originating from classic swine influenza A viruses; the polymerase PB2 (PB2) and polymerase (PA) genes from avian influenza viruses from the North American lineage; and the polymerase PB1 (PB1) gene from human influenza A viruses (Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, 2009).
Eurasian swine lineage
12. The triple-reassortant strain was identified in specimens from patients with infection with triple-reassortant swine influenza viruses before the current epidemic of human infection with S-OIV. HA denotes the hemagglutinin gene, M the M protein gene, NA the neuraminidase gene, NP the nucleoprotein gene, NS the nonstructural protein gene, PA the polymerase PA gene, PB1 the polymerase PB1 gene, and PB2 the polymerase PB2 gene (Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, 2009).
The NA of S-OIV has the closest homology to the Eurasian lineage of swine influenza viruses, such as A/swine/Belgium/1/83 H1N1. Like NA, the M gene of A/California/04/2009 (S-OIV) has the closest homology18 to the M gene in the Eurasian lineage of swine influenza viruses. Analyses of the M gene from all samples from the current epidemic showed a serine (S) 31-to-asparagine mutation [genetic marker (S31N in M2)] that confers resistance to M2 blockers (adamantanes), including amantadine and rimantadine. This phenotype is typical for recent Eurasian lineage swine influenza viruses but has not previously been seen in American swine viruses and adamantane resistance is a characteristic marker of the Eurasian swine lineage. Sequences of the PB1, PB2, PA, NP (replication complex), and NS genes obtained from samples from the current epidemic have the closest homology to the genes in the swine influenza viruses that have been recently isolated in the United States from the North American swine lineage.
The known glycan receptor binding sites (RBS) of the H1 hemagglutinin (HA) protein of S-OIV are typical of many other classical North American swine H1N1 viruses which were isolated recently. Although there are some mutations detected in the HA of the 2009 A(H1N1) viruses that differ from the classical North American swine consensus sequence, none of these were identified in known functionally significant receptor binding sites (Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, 2009). As expected, many of the 2009 A(H1N1) viruses contain amino acid substitutions at putative antigenic sites when compared with seasonal H1 HA (see § 1.10).
A comparison of A/California/04/2009 (or Cal0409) HA with the HA consensus sequences for human-adapted H1N1, avian-adapted H1N1 and swine-adapted H1N1 was accomplished by Soundararajan et al. (2009). This comparison revealed important substitutions in positions 100-300, where the glycan receptor-binding sites (RBS) and antigenic loops are located. Notably, the A/California/04/2009 HA possesses the signature amino acids Asp190 and Asp225 that have been shown to play a key role in conferring specificity to the human α2-6 sialylated glycan receptors (Soundararajan et al., 2009). Amino acids Asp190 and Asp225 are considered to be the “hallmark” amino acids of human-adapted H1N1 HAs that make optimal contacts with the α2-6 glycans (Maines et al. 2009). Therefore, Cal0409 HA is expected to bind with high affinity to α2-6 RBS. Another amino acid substitutions (unique to Cal0409 HA) that have been observed for the first time in human H1N1 HAs by Soundararajan et al. (2009) are at sequence positions 74, 131, 145, 208, 219, 261, 263, 264, 305, 317, 368, 377 and 530. Among these residue positions, 131 and 145 are proximal to the glycan-binding site.
Comparison of the antigenic regions of Cal0409 NA [which is presently the primary target of therapeutic intervention using oseltamivir (Tamiflu)] with the consensus sequences of avian, human and swine-adapted N1 NAs shows that four sequence positions—188, 331, 372 and 402—are novel in the 2009 H1N1 NA. Recently, it has been reported that there is an alarming increase in the oseltamivir (Tamiflu) resistance of H1N1 viruses from 12.3% in 2007-2008 to 98.5% in 2008-2009 season (prior to the outbreak of the 2009 H1N1 infections). Fortunately, the 2009 H1N1 strains were reported to be sensitive to both oseltamivir and zanamivir (Soundararajan et al., 2009).
As the His274Tyr mutation is known to be responsible for resistance of the recent H1N1 human viruses to oseltamivir, Soundararajan et al. (2009) analyzed the potential effect of this mutation, should it occur, on the drug sensitivity of Cal0409 NA. The aforementioned authors proved that the active site has not yet acquired the characteristic mutation His274Tyr that provides resistance to oseltamivir (Tamiflu).
All three of the 20th-century influenza pandemics [1918 (H1N1 subtype), the 1957 “Asian flu” (H2N2), and the 1968 ‘‘Hong Kong flu'' (H3N2)] were caused by viruses containing human adapted PB2 genes (considered critical for the efficient aerosolized respiratory transmission of H1N1 influenza virus), and in general lysine is present at position 627 (Lys627) among the human influenza viruses, whereas a glutamic acid (Glu) is found in this position (627) among the avian influenza isolates that fail to transmit efficiently among ferrets (Maines et al., 2009). In addition, the mutation of this Lys627 to glutamic acid (which is typically found in avian and swine-adapted PB2) in PB2 of the 1918 pandemic strain (SC18) severely reduced its ability to transmit (Soundararajan et al., 2009). Therefore, the best-described marker and determinant of pathogenicity and also of tissue distribution according to Osterhaus et al. (2008) is lysine at position 627 of the polymerase subunit protein PB2 (Salomon and Webster, 2009). Analysis of PB2 in all the 2009 H1N1 strains indicates that it has glutamic acid (Glu) at position 627. On the basis of observation above, it can be concluded that it is expected that the 2009 H1N1 viruses may be capable of transmission between humans, but the efficiency of transmission might be hampered by the absence of Lys627 in PB2 (Soundararajan et al., 2009).
1.10. Seasonal H1N1 Virus
The normal “seasonal” influenza, which occurs predominantly during a six to eight week period in winter, affects between 5-10 % of the population and accounts for 12.000 deaths annually. The majority of its victims are the very young and elderly or those with underlying chest and heart conditions (Honigsbaum, 2008).
The emergence of the novel A/H1N1 (swine-like) influenza virus took place against a background of concurrently circulating seasonal H1N1 and H3N2 influenza viruses (Ginocchio and George, 2009) which their continual presence during seasonal epidemics has remained since 1977. A significant marker of virulence that can be assessed by sequences alone is the degree of identity between the viral hemagglutinin molecules of the new strain (e.g. the 2009 lineage) and those of other human viruses. Low identity indicates antigenically distinct hemagglutinin structures, suggesting that transmission from human tohuman will not be blunted by a degree of “herd” immunity resulting from exposure to similar viruses (Wang and Palese, 2009). The 2009 S-OIV strain is antigenically distinct from and dissimilar to the influenza A (H1N1) strain represented in the 2008-2009 seasonal influenza vaccine (Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, 2009) indicating the presence of the aforementioned low degree of identity between the two virus strains. The 2009 lineage carries three gene segments that share a common (albeit remote) descent from the 1918 “Spanish Influenza” virus with the human seasonal H1N1 virus: segments encoding for nucleocapsid (NP), non-structural (NS), and (perhaps most important) HA proteins (Zimmer and Burke, 2009). A/Brisbane/59/07, which is the current vaccine H1N1 strain and a representative seasonal H1N1 strain, has been associated with severe infectivity due, at least in part, to the fact that its HA and NA have low antigenic cross-reactivity to preexisting humoral immunity (Soundararajan et al., 2009).
Ferret post-infection antisera (Garten et al., 2009) raised against the currently circulating seasonal human A(H1N1) viruses (serum neutralization test using reference antisera raised in animals) did not react with the 2009 A(H1N1) strains. According to Garten et al. (2009), this lack of crossreactivity does not directly equate to a lack of crossprotection in humans between the seasonal A(H1N1) virus and the antigenically distinct and dissimilar 2009 A(H1N1) virus. This is due to the fact that humans have a more complex immune profile than the single-infection used in ferrets for antigenic characterization.
Like seasonal influenza, 2009 H1N1 is thought to be transmissible by 3 routes: contact exposure (when a contaminated hand is exposed to facial membranes), droplet spray exposure (when infectious droplets are projected onto mucous membranes), and airborne exposure (via inhalation of infectious airborne particles). The relative contribution of each of these modes is unknown and likely dependent on such factors as temperature and humidity. The complexities involved with human research to understand influenza transmissibility leave animal studies and mathematical models to inform us.
Annual influenza vaccination is the best public health intervention to prevent human seasonal influenza. Influenza vaccine is available in two trivalent formulations—inactivated and live-attenuated—that contain an A (H1N1), an A (H3N2), and a B virus strain. A semiannual strain selection process is coordinated by the World Health Organization (WHO) to determine the composition of the northern and southern hemisphere influenza vaccines. Updating of the three influenza virus vaccine strains is based on global strain surveillance data (Cox.and Uyeki, 2008). Specifically, the selection of viral strains for inclusion in the vaccine is based on antigenic, genetic and epidemiological data, and practical issues such as the availability of high-growth reassortants suitable for vaccine production (Bush, 2007).
The influenza viruses isolated from mammals are of two classes (Webster, 1999):
1. Those that are closely related to avian virus and appear to have been only transiently established in the mammalian host - these include the transitory introduction into humans of classical swine influenza viruses, which can result in deaths, as occurred at Fort Dix and other isolated introductions
2. Those that have become established in a mammalian host and have diverged progressively from the avian virus ancestor. These include the avian(-like) HIN1 influenza viruses (Eurasian swine) that have become established in pigs in Europe. These viruses have established a permanent lineage in their new host and are evolving rapidly. The established mammalian virus lineages include those isolated from swine and humans. Each of the mammalian virus lineages shows a progressive accumulation of mutations (antigenic drift) and a good correlation of its date of isolation and its position on its respective branch of the phylogenetic tree.
The basic reproductive number (R0) is the number of infections caused by an infected person who is introduced into a completely susceptible population. The estimated R0 for the Fort Dix (1977) swine influenza virus was 1.2. For human pandemic viruses the values of the basic reproductive number (R0) range from 1.8 to 2.0 (Zimmer and Burke, 2009). One of the unusual characteristics of the 2009 A(H1N1) influenza virus as compared with other recent zoonotic influenza viruses is the sustained human-to-human transmission, with basic reproduction ratio (R0) estimates in the range of 1.2 to 1.6, which is higher than that reported for seasonal human influenza A viruses (Munster et al., 2009). A prerequisite for the efficient replication and human-to-human transmission of influenza viruses is that a human-virus-like receptor specificity exists (preferential binding of human pandemic viruses to 6-linked Sia receptors [6' SLN]) [Matrosovich et al., 2008]. However, some virologists are of the view that the current situation of the 2009 Influenza A (H1N1) outbreak is not problematic since this H1N1 lacks the known molecular marker of pathogenicity PB1-F2 (see § 1.4.1), a protein which has been linked to virulence (Kumari et al., 2009). These virologists also suggest that the current H1N1 swine influenza virus causes a mild disease compared to the three major pandemic viruses of the last century (Wang and Palese, 2009). Their last suggestions were confirmed by the fact that the most confirmed cases of novel S-OIV A (H1N1) infection have been characterized by self-limited, uncomplicated febrile respiratory illness and symptoms similar to those of seasonal influenza (a cough, a sore throat, rhinorrhea, headache, and myalgia) [Chang et al., 2009].
Another well-known virulence factor for the influenza virus is the NS1 protein (see § 1.4.1). The C-terminal domain of the NS1 protein contains the ESEV signal in many avian influenza A viruses. The ESEV signal interacts with cellular modulators that contain the PDZ domain. This interaction may increase viral pathogenicity (through the regulation of the activity and trafficking of membrane proteins). Although the NS gene segment of the 2009 S-OIV A (H1N1) originated from an avian virus, it is truncated by a stop codon at position 220 (Chang et al., 2009). This creates a deletion of the PDZ ligand domain, a protein-protein recognition domain involved in a variety of cell-signaling pathways that have been implicated in the pathogenicity of 1918 H1N1 and highly pathogenic H5N1 viruses (Garten et al., 2009). PDZ ligand domains are conserved 80- to 90-amino-acid stretches, often in multiple copies, in proteins associated with the plasma membrane (Perdue, 2008). Hence, NS1 protein in S-OIV does not have the PDZ ligand domain. It is difficult to predict whether a further mutation in humans will change the sequence at position 220 and thereby alter the virulence of S-OIV A (H1N1) [Chang et al., 2009].
The current outbreak of Influenza 2009 A/H1N1 (S-OIV) is a rare recombination of gene segments from swine with avian and human influenza strains. The virulence of the novel S-OIV A (H1N1) may change as the number of adaptive gene mutations increases. Like other influenza A viruses, S-OIV enters host cells by binding to receptors that contain sialic acid (Sia). Adaptive mutations may occur in any other gene segments apart from the receptor binding site (RBS) [no mutation was identified in any functionally significant receptor binding site], and alter viral pathogenesis and virulence. Previously unrecognized molecular determinants are responsible for the ability of the 2009 A(H1N1) virus to replicate and transmit in humans. More studies should be conducted to identify the unrecognized molecular markers which will be used to predict viral virulence and pathogenicity for diagnosis. The novel S-OIV A (H1N1) has inherited sensitivity to oseltamivir (Tamiflu) and resistance to amantadine. The novel 2009 A/H1N1 virus is antigenically distinct to other seasonal flu viruses identified thus far. It has a higher transmissibility as compared to circulating seasonal flu viruses and there appears to be a lack of herd immunity to this agent in the global population.
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 The genomes of the negative-strand viruses can be divided into two types: (a) Nonsegmented genomes for which the first step in replication is transcription of the (-)sense RNA genome by the virion RNA-dependent RNA polymerase to produce monocistronic mRNAs, which also serve as the template for subsequent genome replication. (b) Segmented genomes (Orthomyxoviridae), for which replication occurs in the nucleus, with monocistronic mRNAs for each of the virus genes produced by the virus transcriptase from the full-length virus genome (Cann, 2005).
 Virion is amorphologically complete virus particle.
 Enveloped virions possess an outer (bounding) lipoprotein bilayer membrane.
 They are knob-like structures (spikes) projecting from the surface envelope of a virus particle. They may have hemagglutinating activity and act as cell receptors, or have enzyme activity such as neuraminidase. They are surface antigens (Mahy, 2009).
 Influenza C virus genomes contain seven RNA segments.
 A membrane system in the cell cytoplasm consisting of lamellae and tubules. The site at which biosynthetic processes such as membrane assembly, protein secretion, and organelle biosynthesis occur in eukaryotic cells. The site of maturation of membranes of many viruses (Mahy, 2009).
 Trade name of the drug.
 The addition of adenylate residues, usually to the 3' end of RNA molecules (Mahy, 2009).
 Mechanism by which many viruses induce the “programmed” cell death (a process characterized by cell shrinkage, nuclear condensation, and DNA fragmentation); transforming viruses encode proteins that inhibit cellular apoptotic pathways. Plays a major role in viral infections and in the host response to them (Mahy, 2009).
A set of codons for amino acids uninterrupted by stop codons. Usually encodes one or more proteins (Mahy, 2009).
For optimal translation of an mRNA, positions -3 and +4 relative to the first nucleotide of the initiation codon must be G or A (Mahy, 2009).
 The fluid portion of the cytoplasm outside the organelles (Mahy, 2009).
Cytoplasmic organelle containing hydrolytic enzymes that play an important role in the degradation of material ingested by phagocytosis or endocytosis (Murphy et al., 1999).
hemagglutination inhibition (HI) test which can be used to measure antibody levels (Wagner et al., 2008) and is still the gold standard in antigenic analysis for influenza (Oxford et al., 2003).
A disease temporarily present in an animal community, attacking many animals at the same time, spreading rapidly, and becoming widely diffused (Mahy, 2009).
 As a consequence, infants, still protected by maternal antibody, and persons over the age of 20 years all had neutralizing antibody against swine influenza but not always against human influenza of that period.
Following the transmission to, and independent spread of avian or human influenza A viruses in pigs, these viruses are generally referred to as “avian-like” swine or “human-like” swine, reflecting their previous host (Brown, 2008).
 The degree of relatedness between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules. Hybridization experiments can produce useful information but, for critical analyses, sequence data are needed (Mahy, 2009).
 A diagrammatic representation of the interrelationships between genes. For viruses, it is usually based on genome nucleic acid sequences. The relationship between the common ancestor, intermediate ancestors, and contemporary species are depicted as the root, branches, and leaves of a tree (Mahy, 2009).