Influenza A is an enveloped virus with a negative sense, single-stranded, segmented RNA genome belonging to the family of Orthomyxoviridae. It appears most abundantly in a roughly spherical shape with a diameter of 80 - 120 nm, but also filamentous particles can be observed.
Subtypes of influenza viruses are classified according to their surface glycoproteins, the hemag-glutinin (HA) and the neuraminidase (NA). The nomenclature is composed of information on the virus type, species from which it has been isolated (only if it has not been isolated from humans), isolate number and year of isolation. For IAV the HA and NA subtypes are also added (e.g. A/Hong Kong/2007/99 (H3N2)).
3.1.1 Structure and replication cycle of influenza A viruses
The virion of the IAV is covered with spikes of the two surface glycoproteins HA and NA whereby HA is incorporated four times more often than NA (Palese and Shaw, 2007). Addi-tionally, matrix ion channel (M2) proteins are present in the host cell-derived lipid membrane.
The space between the envelope and the virion core is filled with a matrix of M1 proteins.
Each of the eight viral RNA gene segments is encapsidated by nucleoprotein (NP) in associa-tion with the heterotrimeric RNA-dependent RNA polymerase (consisting of PB1, PB2 and PA). This gene / protein complex is called the viral ribonucleoprotein (vRNP). Next to the vRNP the nuclear export protein (NEP; alternatively: NS2) is present in the core structure of IAVs (Figure 3.1.1.1).
The eight viral RNA (vRNA) gene segments are numbered according to their length in a descending order. PB2, HA, NP and NA are each encoded by an entire segment, segment 1,
1 INTRODUCTION
Figure 3.1.1.1: Structure of influenza A viruses
Scheme of the structure of IAVs with segmented RNA genome and viral proteins (Source: Nelson and Holmes (2007))
4, 5 and 6, respectively. Next to the primary product, PB1, two additional proteins, PB1-F2 (Chen et al., 2001) and PB1-N40 (Wise et al., 2009) are encoded on segment 2. However, PB1-F2 which is generated through a frame shift is not present in all IAVs (Chen et al., 2001). Recently, it has been shown that segment 3 which encodes for a subunit of the viral RNA-dependent RNA polymerase (PA) contains a second open reading frame that codes for PA-X, another viral protein (Jagger et al., 2012). Segment 7 codes for the M1 matrix protein and can additionally express the M2 ion channel utilizing the RNA splicing machinery (Lamb et al., 1981). Finally, mRNA splicing makes it possible that segment 8 can encode the two viral proteins, NS1 and NEP / NS2 (Lamb et al., 1980).
The viral replication cycle (Figure 3.1.1.2) is initiated by the recognition and binding of N-acetylneuraminic (sialic) acid on the surface of the host cell by cleaved HA (Skehel and Wiley, 2000).
IAVs show a preferential specificity for either α2,3- orα2,6-linked sialic acids which implies that they can infect each host cell that expresses one of these sialic acids but with variable efficiency (Couceiro et al., 1993). Cleaved HA consists of HA1 which contains the receptor binding and antigenic sites and HA2 which is important for the fusion of the virus enve-lope with the host cell membrane. The attached viral particle enters the host cell through receptor-mediated endocytosis. The resulting endosome exhibits a low pH of 5 to 6 leading to a conformational change in the HA. Afterwards, the fusion peptide mediates merging of the
Figure 3.1.1.2: Scheme of the influenza A virus replication cycle
After receptor-mediated attachment of the virion to the host cell surface viral particles are endocytosed. (1) Viral RNA segments are released into the cytoplasm and transported to the nucleus. (2) In the nucleus mRNA synthesis and RNA replication take place. (3) mRNAs are exported into the cytoplasm where they are translated. (4) Viral proteins that are needed for replication and transcription are transported back into the nucleus in the early stage of infection. (5) During the late phase of infection M1 and NS2 enable nuclear export of newly synthesized vRNPs (6). (7) All viral components are transported to the cell membrane. (8) The replication cycle ends with budding of progeny virions from the host cell membrane.
Figure modified from (Neumann et al., 2009).
viral envelope with the endosomal membrane. Additionally, M2 ion channel proteins acidify the inner core of the virion and vRNPs are separated from the viral matrix. Thus, vRNPs are released into the cytoplasm of the host cell (reviewed in: Sieczkarski and Whittaker (2005)).
All proteins of the vRNP (NP, PA, PB1 and PB2) exhibit nuclear localization signals (NLSs) enabling the vRNP and other viral proteins enter the host cell nucleus (Cros and Palese, 2003; Boulo et al., 2007). There, the RNA-dependent RNA polymerase complex uses the negative-sense vRNA as template to generate two different kinds of RNAs. Complementary RNA (cRNA) is transcribed to produce genomic vRNA and viral messenger RNA (mRNA) is produced for the synthesis of viral proteins. Viral mRNAs need to be polyadenylated and 5’-capped in order to be translated by the host cell. In contrast to host cell mRNAs, they receive their polyadenylation signal through a stretch of five to seven uracil residues encoded in the vRNA that transcribes to a poly(A) tail (Li and Palese, 1994). The 5’-cap is achieved through a mechanism called ’cap-snatching’ in which PB1 and PB2 proteins hijack pre-mRNA of the host cell (Krug, 1981). Export of the viral RNP and other viral proteins can occur after acquiring the poly(A) signal and the 5’ cap. Viral proteins are synthesized on host ribosomes in the cytoplasm. M1, NP and NS1 are transported back into the nucleus.
Other proteins, HA, NA and M2, are transported to and integrated into the host cell mem-brane. Packaging of the virion is mediated through M1 that brings the RNP-NEP complex
1 INTRODUCTION
near to the three membrane-bound proteins (Palese and Shaw, 2007). Specific packaging signals are responsible to ensure a complete viral genome in most of the virions (Hutchinson et al., 2010). Virus particles are released on the apical side of polarized cells (Nayak et al., 2009). Budding of the progeny virions is possible when NA has cleaved terminal sialic acid residues from glycoproteins on the cell surface. The last important step for newly generated influenza virions is the cleavage of the HA by host proteases otherwise they will not be able to infect other host cells (Klenk et al., 1975; Lazarowitz and Choppin, 1975).
3.1.2 Influenza A - a zoonotic pathogen
Even though ’the flu’ is mainly known to cause annual re-occuring seasonal epidemics in hu-mans, IAVs are also able to infect plenty of homeothermic vertebrate species. This includes many domesticated animals like swine, poultry and horses (Landolt and Olsen, 2007) which receive most attention due to the implications for public health and economy. However, also other species such as minks, felids, dogs and marine mammals can get infected (Figure 3.1.2.1). In contrast to humans, wild birds can be infected with many influenza A subtypes, but most infections are asymptomatic.
Figure 3.1.2.1: Host range of different influenza A virus subtypes
Wild aquatic birds are the primary reservoir of IAVs and can be infected with each IAV subtype. IAV subtypes that are able to infect poultry, pigs, humans, marine animals, horses and dogs among others are indicated.
(Source: Kalthoff et al. (2010)).
For ducks, it has been shown that low pathogenic avian influenza viruses (LPAI) replicate primarily in cells lining the intestinal tract where virus can accumulate to high concentrations and subsequently be excreted (Webster et al., 1978). This qualifies birds, especially wild migratory waterfowl and shorebirds, as the natural reservoir for influenza A viruses (Webster et al., 1992). The HA cleavage site needs to be polybasic to convert a LPAI with a monobasic
cleavage site into a highly pathogenic avian influenza virus (HPAI). Thereafter, IAV has the possibility to go systemically, leading to a mortality of up to 100% in poultry (de Wit and Fouchier, 2008).
IAVs exhibit zoonotic potential because of their high genetic variability. Transmission of IAV into a new host species occurs through a combination of ’antigenic shift’ and ’antigenic drift’.
New subtypes with changed pathogenicity can arise through ’antigenic shift’ via reassortment of different IAV subtypes infecting the same host cell. Mutations caused by the missing proofreading function of the viral polymerase occur in each replication cycle which leads to the so called ’antigenic drift’ (Webster et al., 1992). Recently, IAVs have also been found in bats. These viruses belong to new subtypes (H17N10 and H18N11) and are evolutionarily separated from all other strains that are currently circulating in other species (Tong et al., 2012). The addition of bats to the list of possible hosts for IAVs dramatically increases the host range because bats are representing ∼ 20% of all known mammals (Mehle, 2014). In summary, it is important that we consider IAV infections as a ’One Health’ problem which means that researchers from different fields of expertise (e.g. veterinarians, virologists and pharmacists) need to work together in order to be prepared or even prevent the next IAV pandemic.
3.2 Contribution of viral and host factors on the pathogenesis of influenza virus