University of Veterinary Medicine Hannover Institute of Virology
Use of ex vivo and in vitro cultures of differentiated airway epithelial cells to analyze the infection by influenza A viruses
INAUGURAL – DISSERTATION
In partial fulfilment of the requirements of the degree for Doctor of Natural Sciences
- Doctor rerum naturalium - (Dr. rer. nat)
Submitted by Yuguang Fu Henan province, China
Hannover 2018
Institute of Virology
University of Veterinary Medicine Hannover
Prof. Dr. Peter Valentin-Weigand Institute of Microbiology
University of Veterinary Medicine Hannover
1. Supervisor Prof. Dr. Georg Herrler Institute of Virology
University of Veterinary Medicine Hannover
Prof. Dr. Peter Valentin-Weigand Institute of Microbiology
University of Veterinary Medicine Hannover
2. Supervisor Prof. Dr. Maren von Köckritz-Blickwede Institute of Biochemistry
University of Veterinary Medicine Hannover
Data of examination: 25.10.2018
Fu Y, Tong J, Meng F, Hoeltig D, Liu G, Yin X, Herrler G. Ciliostasis of airway epithelial cells facilitates influenza A virus infection. Vet Res. 2018, 49(1):65.
Yuguang Fu, Ralf Dürrwald, Fandan Meng, Jie Tong, Nai-Huei Wu , Ang Su, Xiangping Yin, Ludwig Haas, Michaela Schmidtke, Roland Zell, Andi Krumbholz, Georg Herrler. Infection studies in pigs and airway epithelial cells reveal an evolution of A(H1N1)pdm09 influenza A viruses towards lower virulence.
Submitted
Poster presentation:
13-14/10/2016 National Symposium on Zoonoses Research 2016, Berlin, Germany Importance of the ciliary activity of the airway epithelium in preventing influenza virus infection.
Yuguang F., Zhenhui S., Fandan M. and Georg H.
19-22/10/2016 6th European Congress of Virology, Hamburg, Germany
Importance of the ciliary activity of the airway epithelium in preventing influenza virus infection.
Yuguang F., Zhenhui S., Fandan M. and Georg H.
22-25/03/2017 27th Annual Meeting of the Society for Virology, Marburg, Germany Importance of the ciliary activity of the airway epithelium in preventing influenza virus infection.
Fu Y., Meng F. and Herrler G.
LIST OF ABBREVIATIONS ... I ABBREVIATIONS FOR AMINO ACIDS ... III ABSTRACT ... IV ZUSAMMENFASSUNG... VI
INTRODUCTION ... 1
1. Influenza A viruses ... 1
1.1 Disease ... 1
1.2 Taxonomy ... 2
1.3 Structure and life cycle ... 3
1.4 Proteins ... 4
2. Genetic variation of influenza A viruses ... 11
2.1 Point mutations ... 11
2.2 Reassortment events... 12
3. Pigs and swine influenza A viruses ... 13
3.1 Pigs ... 13
3.2 Swine influenza A viruses ... 13
4. Pandemics ... 16
4.1 Spanish influenza pandemic in 1918... 16
4.2 The Asian influenza pandemic in 1957 ... 17
4.3 The Hong Kong influenza pandemic in 1968 ... 17
4.4 The Russian influenza pandemicn in 1977 ... 17
4.5 The Swine-origin influenza pandemic in 2009 ... 17
5. Barrier function of the respiratory tract ... 19
5.1 Mucociliary clearance system ... 19
5.2 Intercellular junction complexes ... 20
5.3 Antimicrobial peptides ... 20
6. Primary culture systems of differentiated airway epithelial cells ... 22
6.1 Precision-cut lung slices ... 22
6.2 Air-liquid interface cultures ... 23
7. Infection of pigs by five A(H1N1)pdm09 viruses ... 24
7.1 Dyspnea and rectal temperature of animals infected with A(H1N1)pdm09 viruses ... 24
7.2 Lung lesions and viral load in the lung of pigs infected with A(H1N1)pdm09 viruses ... 27
8. Aim of the study ... 30
MATERIAL AND METHODS ... 31
1. Cells and viruses ... 31
2. Preparation of porcine differentiated airway epithelial cells ... 31
2.1 Precision-cut lung slices ... 31
2.2 Air-liquid interface cultures ... 32
3. Effects of hypertonic salt on ciliary activity of PCLS ... 33
4. Effect of 2% NaCl on the infectivity of H3N2 type virus ... 33
5. Virus infection and titration ... 34
6. Effects of A(H1N1)pdm09 viruses on ALI cultures ... 34
7. Immunofluorescence assay ... 35
8. Statistical analyses ... 36
RESULTS ... 37
1. Ciliostasis of airway epithelial cells facilitates influenza A virus infection ... 37
1.1 Reversible ciliostasis induced by hypertonic salt ... 37
1.2 Effect of 2% NaCl on the infectivity of influenza A virus ... 39
1.3 Infection of PCLS under ciliostatic condition ... 40
2. In vitro studies in porcine differentiated airway epithelial cells reveal an evolution of human-derived A(H1N1)pdm09 viruses towards lower virulence between 2009 and 2015 ... 41
2.1 Infection of well-differentiated airway epithelial cells ... 41
2.2 Tissue tropism of the viruses ... 41
2.3 Effect of virus-infection on trans-epithelial electrical resistance ... 42
2.4 Replication kinetics of the viruses in well-differentiated PBEC ... 44
2.5 Relative cilia coverage of epithelial cells after infection by A(H1N1)pdm09 ... 44
2.6 Relative thickness of ALI cultures infected with A(H1N1)pdm09 viruses at 8 days post infection ... 47
DISCUSSION ... 49
1. Ciliostasis of airway epithelial cells facilitates influenza A virus infection ... 49
2. In vitro studies in porcine differentiated airway epithelial cells reveal an evolution of human-derived A(H1N1)pdm09 viruses towards lower virulence between 2009 and 2015 ... 51
3. Summary and outlook ... 56
RERERENCE ... 58
ACKNOWLEDGEMENT ... 77
LIST OF ABBREVIATIONS aa Amino acid residue
ALI Air-liquid interface cultures BSA Bovine serum albumin CO2 Carbon dioxide
Cy3 Indocarbocyanine
e.g. Exempli gratia (for example) EMEM Eagle’s minimal essential medium
Fig Figure
FITC Fluorescein isothiocyanate
g Gramm or gravity
h Hour
HA Hemagglutinin
hpi Hours post infection
M Matrix protein
MDCK Madin-Darby canine kidney
Min Minute
NA Neuraminidase
NP Nucleoprotein
NS Non-structural protein NS1 Non-structural protein 1
NS2 Non-structural protein 2, Nuclear export protein PA Polymerase acidic protein
PB1 Polymerase basic protein 1 PB2 Polymerase basic protein 2 PBS Phosphate-buffered saline PCLS Precision-cut ling slices RBS Receptor binding site RNP Ribonucleoprotein
TCID50 Tissue culture infective dose TEER Transepithelial electrical resistance WHO World Health Organization
oC Degree Celsuis
μl Microliter
μm Micrometer
ABBREVIATIONS FOR AMINO ACIDS
Animo acids 3-letter symbol 1-letter symbol
Alanine Ala A
Arginine Arg R
Asparagine Asn N
Asparitc acid Asp D
Cysteine Cys C
Glutamic acid Glu E
Glutamine Gln Q
Glycine Gly G
Histidine His H
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
ABSTRACT
“Use of ex vivo and in vitro cultures of differentiated airway epithelial cells to analyze the infection by influenza A viruses”
Influenza A viruses are common pathogens in the respiratory tract of many mammalian and avian species. Porcine airway epithelial cells are susceptible to infection not only by swine influenza viruses but also by human and avian influenza A viruses. Therefore, pigs may play an important role in the interspecies transmission and have been described as “mixing vessel”. To analyze the infection of influenza A viruses in the actual target cells of the respiratory tract, we applied two culture systems for differentiated airway epithelial cells: precision-cut lung slices (PCLS) and air-liquid interface (ALI) cultures that provide a suitable ex vivo and in vitro tool to analyze the interaction of influenza viruses with epithelial cells of the respiratory tract.
To analyze whether the ciliary activity of ciliated cells can impede the infection by influenza A viruses, porcine PCLS were infected with a swine influenza A virus of the H3N2 subtype in the presence or absence of ciliary activity. During the viral attachment step, the infectious medium was supplemented with 2% NaCl which causes reversible ciliostasis. Viral titers were 2- or 3- fold higher at 24 h or 48 h post-infection when cells were infected under ciliostatic conditions compared to infection of slices with ciliary activity. This result indicates that the ciliary beating not only transports the mucus out of the respiratory tract, it also impedes virus infection.
To determine the virulence of A(H1N1)pdm09 influenza viruses, strains isolated in the years following the 2009 influenza pandemic were selected to infect ALI cultures of differentiated airway epithelial cells. Four strains isolated from human patients in 2009 (2x), 2010, 2015, and one strain isolated from a pig in 2014 were applied. In the in vitro experiments with ALI cultures, three parameters were used to determine the virulence of the viruses; (i) the amount of virus released into the supernatant, (ii) the loss of ciliated cells, (iii) reduction of the thickness of the epithelial cells layer. The results indicated that the virulence of viruses of 2014/2015 was significantly lower than that of viruses from 2009/2010. A similar result was obtained by a collaboration
partner who compared the virulence of the five virus strains in infection trials with pigs. The viruses from 2009/2010 have 20 amino acid exchanges in viral proteins in common that distinguish them from the viruses of 2014/2015. Our results suggest that A(H1N1)pdm09 influenza viruses have undergone an adaptation process in the years following the 2009 pandemic, and this adaptation is associated with a loss of virulence. The ALI culture is a promising tool to predict the virulence of influenza viruses which may help in the future to replace animal experiments.
ZUSAMMENFASSUNG
“Anwendung von ex vivo- und in vitro-Kulturen differenzierter porziner Atemwegsepithelzellen für die Untersuchung der Infektion durch Influenzaviren”
Influenza-A-Viren sind häufige Krankheitserreger im Respirationstrakt vieler Säugetiere und Vögel. Atemwegsepithelzellen von Schweinen sind nicht nur empfänglich für die Infektion durch porzine Influenzaviren, sondern auch für die Infektion durch humane und aviäre Influenzaviren. Deshalb können Schweine eine wichtige Rolle spielen bei der Interspezies-Übertragung und werden als
„Mischgefäß“ bezeichnet. Um die Infektion durch Influenza-A-Viren in den eigentlichen Zielzellen im Respirationstrakt zu untersuchen, haben wir zwei Kultursysteme für differenzierte Atemwegsepithelzellen zur Anwendung gebracht:
Präzisionslungenschnitte (precision-cut lung slices, PCLS) und “air-liquid-interface”
(ALI)-Kulturen, die geeignete ex vivo- und in vitro-Systeme sind, um die Interaktion von Influenzaviren mit Epithelzellen des Respirationstrakts zu untersuchen.
Um zu untersuchen, ob die Zilienaktivität der zilientragenden Zellen die Infektion durch Influenza-A-Viren behindern kann, wurden PCLS mit einem Schweineinfluenzavirus des Subtpys H3N2 infiziert, und zwar sowohl in der Gegenwart als auch in der Abwesenheit der Zilienbewegung. Für die Unterbindung der Zilienaktivität wurde dem Medium während des viralen Anheftungsprozesses 2%
NaCl zugesetzt, wodurch eine reversible Ziliostase ausgelöst wird. Die Virustiter im Zellüberstand waren 24 h oder 48 h nach der Infektion zwei bzw. dreifach höher, wenn die Zellen unter ziliostatischen Bedingungen infiziert wurden im Vergleich zu einer Infektion von Zellen mit Zilienaktivität. Dieses Ergebnis zeigt, dass die Zilienaktivität nicht nur den Mukus aus dem Respirationstrakt heraustransportiert, sondern auch die Virusinfektion behindert.
Um die Virulenz von A(H1N1)pdm09 Influenza-A-Viren zu vergleichen, wurden Stämme von Influenza-A-Viren die in den Jahren nach der Influenza-Pandemie von 2009 isoliert worden waren, ausgewählt, um ALI-Kulturen differenzierter porziner Atemwegsepithelzellen zu infizieren. Vier Stämme von menschlichen Patienten waren in den Jahren 2009 (2x), 2010 und 2015 isoliert worden, ein Stamm war 2014 von
einem Schwein isoliert worden. In den in vitro-Experimenten mit ALI-Kulturen wurden drei Parameter verwendet, um die Virulenz zu bestimmen: (i) die Menge der in den Zellüberstand freigestzten infektiösen Viren, (ii) der Verlust zilientragender Zellen, (iii) die Reduktion der Dicke der Epithelzellschicht. Die Ergebnisse zeigen, dass die Virulenz der Viren von 2014/2015 signifikant niedriger war als jene der Viren von 2009/2010. Ein ähnliches Ergebnis wurde von einem Kollaborationspartner erzielt, der die Virulenz der fünf Virusstämme in Infektionsversuchen mit Schweinen verglich. Die Viren von 2009‘/2010 haben 20 Aminosäureaustausche gemeinsam, durch die sie sich von den Viren von 2014/2015 unterscheiden. Unsere Ergebnisse sprechen dafür, dass A(H1N1)pdm09 Influenzaviren in den Jahren nach der Pandemie von 2009 einen Adaptationsprozess durchgemacht haben. Diese Adaptation ist verbunden mit einem Verlust der Virulenz. ALI-Kulturen sind ein vielversprechendes Werkzeug um die Virulenz von Influenzaviren vorherzusagen. In der Zukunft kann es dazu beitragen, Tierversuche zu ersetzen.
INTRODUCTION 1. Influenza A viruses 1.1 Disease
Influenza is a common disease in the respiratory tract caused by influenza viruses in humans and animals. Due to frequent epidemics and occasional pandemics, it is a global health risk (Samy and Lim, 2015). This disease may occur throughout the seasons and affects mainly children, old people and people with low resistance and immunity (Nicholson et al., 2003). Influenza viruses are divided into three different types (influenza A, B and C) on the basis of the antigenic relationship of the nucleoproteins (NP) and matrix (M) proteins (Lamb and Choppin, 1983; Lee and Saif, 2009; Spanakis et al., 2014). They are responsible for substantial economic losses due to hospitalizations, treatment and prevention. The majority of influenza cases reported worldwide are caused by influenza A viruses which can infect a wide range of hosts including humans, pigs, poultry, wild birds, ferrets and horses (Jagger et al., 2012).
Except humans, seals and ferrets are susceptible to influenza B virus infection, pigs and dogs can be infected by influenza C virus (Jakeman et al., 1994; Matsuzaki et al., 2002; Osterhaus et al., 2000; Taubenberger et al., 2008). Influenza disease in humans caused by seasonal influenza viruses may range from mild to severe symptoms or even to death, and is characterized by headache, sore throat, cough, fever and severe malaise. About 3 to 5 million severe cases infected by seasonal influenza viruses each year in the world have been estimated by the World Health Organization (WHO), the United States Centers for Disease Control and Prevention (US-CDC) and global health partners, and about 650 000 cases of death associated with seasonal influenza (http://www.who.int/en/news-room/detail/14-12-2017-up-to-650-000-people-die-of-re spiratory-diseases-linked-to-seasonal-flu-each-year). Historically, there were several pandemic influenza outbreaks caused by influenza A virus in humans including the Spanish influenza (H1N1) in 1918, the Asian influenza (H2N2) in 1957, the Hong Kong influenza (H3N2) in 1968, the Russian influenza (H1N1) in 1977 and the swine-origin influenza (H1N1) in 2009 (Horimoto and Kawaoka, 2001; Horimoto and Kawaoka, 2005; Kilbourne, 2006; Presanis et al., 2009). The Spanish influenza occurred in 1918 and was the most severe pandemic influenza in the history which
killed approximately 50 million people worldwide (John and Muller, 2002). The latest swine-origin pandemic influenza occurred in 2009 which caused about 150 000 to 570 000 humans deaths in the world during the first 12 months since its outbreak (Dawood et al., 2012; Garten et al., 2009; WHO 2010). In pig populations, influenza is an acute respiratory disease characterized by coughing, labored breathing, fever, loss of appetite that result in slow growth. The morbidity rates of the influenza disease in pigs are high and can reach 100%, but the mortality rates are low (Kyriakis CS, 2013).
In poultry industry, influenza of chickens, turkeys and other birds infected by highly pathogenic avian influenza virus strains of the H5N1 and H7N9 subtypes results in acute clinical disease with high mortality and morbidity. Occasionally, some subtypes of influenza A viruses can overcome species barriers to infect a new host. For example, the H5N1 subtype of highly pathogenic avian influenza (HPAI) viruses infected humans in Hong Kong in 1997, and caused human cases and deaths in 2003 and 2004 due to its re-emerging; an outbreak of the H7N9 subtype of HPAI was reported in China in 2013 (Class et al., 1998; Dortmans et al., 2013; Li et al., 2004; Yu et al., 2013). A new reasserted swine-origin H1N1 subtype of influenza A virus caused a pandemic in 2009 (Gabriele et al., 2009).
1.2 Taxonomy
Influenza A viruses belong to the genus Influenzavirus A within the family Orthomyxoviridae which comprises another five genera: Influenzavirus B, Influenzavirus C, Thogotovirus, Quaranjavirus and Isavirus (Presti et al., 2009).
Based on the antigenic relationship between hemagglutinin (HA) and neuraminidase (NA), two important glycoproteins on the viral surface, influenza A viruses are classified into different subtypes (Lamb and Choppin, 1983). So far, 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11) have been recognized. Among them, except for the H17N10 subtype which has been isolated from yellow-shouldered bats and for the H18N11subtype isolated from flat-faced fruit bats, all other subtypes (H1-H16 and N1-N9) of influenza A viruses have been detected in waterfowl which are the major natural reservoir for influenza A viruses (Mehle, 2014; Taubenberger and Kash, 2010;
Tong et al., 2012; Tong et al., 2013). Isolates of influenza A viruses are designated as follows: A/species/region/number/year (H- and N-), e.g. A/swine/Schlallern/2014
(H1N1). After the viral type (A, B, or C) the host species is indicated, from which the virus was isolated (if viruses come from humans, the host designation is omitted); this information is followed by the geographic origin (where the virus was isolated), the strain number, the year of the virus isolation, and the HA and NA subtypes.
1.3 Structure and life cycle
Influenza A viruses (Fig.1) have a spherical shape with a diameter of 80-120 nm, they are enveloped with a lipid membrane derived from the plasma membrane of the infected cell. There are three proteins, HA, NA, and matrix 2 protein (M2, ion channel protein), embedded in this membrane. Below the lipid membrane is the matrix protein 1 (M1) which determines the shape of influenza A viruses and interacts with the cytoplasmic domains of HA, NA and M2 proteins, and thus mediates a connection with the ribonucleoproteins (RNPs). The genome of influenza A viruses is composed of eight segments of single-stranded, negative-sense RNA, about 13 kb in length, and encodes at least 13 proteins including HA, NA, M1, M2, nucleoprotein (NP), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), PB1-F2 protein, nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), also named as nuclear export protein (NEP), as well as two new proteins PA-X and PB1-N40 which are synthesized from PA mRNA and PB1 mRNA, respectively (Bouvier and Palese, 2008; Chen et al.,2001; Muramoto et al, 2013;
Vasin et al., 2014). Among these proteins, HA, NA, M1, M2, NP, PA, PB1 and PB2 are structural proteins, NS1, NS2 (NEP), PB1-F2, PA-X and PB1-40 are accessory proteins (Goraya et al., 2015; Jagger et al., 2012).
Fig. 1 Schematic diagram of an influenza A virus (Wendel et al., 2015)
The life cycle (Fig. 2) of influenza A viruses is initiated by the interaction of the HA protein and cellular receptors on the surface of target cells. During this process, HA mediates the binding to its receptor, and the fusion of viral and cellular membranes which results in viral ribonucleoproteins (vRNPs) released into the cytoplasm. Then vRNPs are transported into the nucleus where the replication of the viral genome and the transcription of messenger RNAs (mRNA) is carried out by PA, PB1, PB2, NP and PB1-F2 proteins. Proteins required for transcription and replication are translated after mRNAs have been transported to the cytoplasm and imported back to the nucleus. The M1 protein and NEP protein export newly synthesized vRNPs from the nucleus to the cytoplasm. Virions are assembled at the plasma membrane and released from infected cells with the help of the NA protein which releases sialic acids and thus inactivates virus receptors on the cell surface.
Fig. 2 Schematic diagram of the influenza viral life cycle (Zheng et al., 2013) 1.4 Proteins
1.4.1 Hemagglutinin
The HA protein is an important protein in the viral membrane, encoded by segment 4 and folded as a rod-shaped trimer. HA protein occurs in 18 subtypes. H17 and H18 subtypes are exceptional as they belong to bat viruses; other subtypes are divided into
two groups on the basis of the sequence and structural properties. H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16 subtypes belong to group 1, group 2 consists of H3, H4, H7, H10, H14 and H15 subtypes (Russell et al., 2004). The HA protein determines the host tropism of influenza A viruses by interaction with virus receptors (sialic acid-containing macromolecules) expressed on the cellular surface of target cells.
(Chen et al., 1998; Goraya et al., 2015; Lamb and Choppin, 1983; Skehel and Wiley, 2000; Wilson et al., 1981). The HAs of human and swine influenza A viruses preferentially bind to α-2,6-linked sialic acid, whereas avian influenza A viruses have a preference to recognize α-2,3-linked sialic acid. This property is consistent with α-2,6-linked sialic acid being predominant on epithelial cells in the human and swine airways and α-2,3-linked sialic acid being present on epithelial cells in the intestinal tract of birds (Ito et al., 1998). The binding specificity of influenza A viruses is determined by some domains within the receptor binding site of HA: the 130-loop, the 220-loop and the 190-α helix (Weis et al., 1988; Wilson et al., 1981). Some mutations of amino acid residues in these domains may result in the change of the binding specificity. For example, Gln and Gly residues at position 226 and 228 of H2 and H3 HA subtypes are associated with a preferential binding to α-2,3-linked sialic acid, whereas Leu and Ser residues at these two positions determine a preference for α-2,6-linked sialic acid. For the H1 subtype, Asp residues at position 190 and 225 confer preferential binding to α-2,6-linked sialic acid, whereas Glu and Gly at these positions recognize α-2,3-linked sialic acid preferentially (Steven et al., 2004).
Furthermore, the receptor binding site of HA protein has antigenic properties and can induce the infected host to generate protective antibodies against the corresponding virus subtype by blocking virus attachment (Mallajosyula et al., 2014).
In addition to binding to the receptors and determining the binding specificity and affinity, HA mediates the fusion between the viral and cellular membranes to release viral RNP into infected cells. This fusion function is performed by the HA2 polypeptide which is activated after HA cleavage by proteases. Under low pH-conditions, the cleaved HA undergoes an irreversible change which brings the N-terminus of the activated HA2 polypeptide into a position to mediate the fusion of viral and cellular membrane (Carr and Kim, 1993; Skehel and wiley, 2000; Weber et
al., 1994; Wharton et al., 1995). The cleavage efficiency is determined by the available proteases and the amino acid sequence of the cleavage site. In this way, HA plays a crucial role in the viral virulence, tropism and pathogenicity (Garten and Klenk, 1999; Skehel and wiley, 2000). Highly pathogenic influenza viruses have a polybasic sequence R-X-R/K-R at the cleavage site of HA. This sequence can be recognized specifically and cleaved efficiently by ubiquitous proteases (furin-like enzymes), which are distributed in a wide range of tissues, resulting in systemic infection and systemic symptoms (Bottcher-Friebertsgauser et al., 2013; Suguitan et al., 2012). In contrast, low-pathogenic influenza viruses have a monobasic sequence (single Arg residue) Q/E-X-R at the cleavage site of HA. This sequence can be recognized and cleaved efficiently by trypsin-like enzymes, which are present on epithelial cells of the respiratory tract and/or intestinal tract, and lead to local infections. A change in the cleavage site and thus in the cleavability of HA is crucial for the change of influenza A viruses from low-virulence to highly virulent viruses (Horimoto and Kawaoka, 2005; Hirst et al., 2004).
1.4.2 Neuraminidase
The NA protein is encoded by segment 6 and folded as a mushroom-shaped tetramer.
Each monomer is composed of three domains: a membrane anchor, a rod-shaped stalk region, and a globular head which possesses the enzyme site, respectively (Air, 2012;
Gamblin and Skehel, 2010). The catalytic site within the globular head domain is lined by some conserved amino acids: Arg118, Asp151 and others (Colman et al., 1989). The globular head domain includes not only the enzyme site, but also a calcium binding site (Bossart-whitaker et al., 1993; Tulip et al., 199). Except N10 and N11 subtypes discovered recently in bats (Tong et al., 2012), N1-N9 subtypes of influenza A viruses NA are divided into two groups. N1, N4, N5 and N8 subtypes belong to group 1, and group 2 includes N2, N3, N6, N7 and N9 subtypes. On the viral surface, the NA protein is surrounded by the most predominant protein HA (Harris et al., 2006). During the viral life cycle, NA protein contributes three function:
(i) helping the virus to approach the epithelial cell surface of the respiratory tract by desialylation of inhibitory mucins, (ii) facilitating the release of newly synthesized virions from infected cells by releasing sialic acid from cell surface receptors and (iii)
preventing the aggregation of viral particles by removing sialic acids from the viral surface (Matrosovish et al., 2004; Wagner et al., 2000; Wagner et al., 2002). The amount of NA protein on the viral surface is much lower compared to that of the HA protein, but antigenic epitopes surrounding the enzyme site within the globular head domain can trigger the host to produce antibodies that prevent influenza A virus infections (Air, 2012; Air et al., 1990).
1.4.3 Matrix protein
Matrix protein M1 of influenza A virus together with the M2 protein are encoded by segment 7 (Horimoto and Kawaoka, 2001; Webster et al., 1992). These two proteins are important for assembly and morphology of influenza A virus (Liu et al., 2017).
The M1 protein is the most abundant protein in the viral particle, and forms M1-M1 interphases at the inner side of the membrane which determine the viral structure at different stages of the life cycle depending on the pH value and which affect the viral budding (Harris et al., 2001; Safo et al., 2014). M1 protein has 252 amino acid residues and comprises three domains: the N-terminal domain, the middle domain and the C-terminal domain. The M1 protein not only interacts with HA and NA, but also binds to newly synthesized RNP to transport vRNPs from the nucleus to the cytoplasm with the help of NS2 for packaging in new virions (Ali et al., 2000; Cao et al., 2012; Jin et al., 1997; Noton et al., 2007; Ye et al., 1999; Zhang et al., 200). M2 protein has 97 amino acid residues folded as tetramers and is the third protein on the viral surface. As an ion channel, M2 is crucial for the viral RNP release into the cytoplasm by eliciting viral uncoating during the viral entry process (Edinger et al., 2014). In addition to the effect on viral morphology and infectivity, M2 protein also affects the interaction between NP and RNA during virus assembly (Chen et al., 2008;
Iwatsuki-Horimoto et al., 2006; McCown and Pekosz, 2005; McCown and Pekosz, 2006; Rossman et al., 2010).
1.4.4 Non-structural protein
Non-structural proteins of influenza A virus include two proteins: NS1 and NS2 that are encoded by segment 8 and translated by viral mRNA generated by splicing. The NS1 proteins are expressed in the nucleus and cytoplasm of infected cells, possess an amino acid length ranging from 215 to 237 and include three domains: an N-terminal
RNA-binding domain, a central effector domain and a C-terminal region (Cheng et al., 2018; Hale et al., 2008; Krug, 2015). Based on these domains, NS1 protein contribute several functions during the life cycle of influenza A viruses: they (i) inhibit the transcription and translation of host mRNA, (ii) counteract the innate immunity response (antagonist to type I interferon of host) and (iii) promote the translation of viral mRNA (Fortis et al., 1994; Gack et al., 2009; Garcia-Sastre, 2001; Guo et al., 2007).
The NS2 (NEP) protein is a polypeptide comprising 121 amino acids (Lam and Lai, 1980). At the beginning, this small protein was named as nonstructural protein because it was thought to have no structural function. Subsequently, this protein was found to be present in virions interacting with M1, and demonstrated to have the function to export vRNPs from the nucleus to the cytoplasm in infected cells, to guarantee the packaging of viral genome into new virions on the cellular surface (O'Neill et al., 1998; Richardson and Akkina, 1991; Ward et al., 1995; Yasuda et al., 1993;). Therefore, this protein was renamed as NEP. Apart from export of vRNPs from the nucleus to the cytoplasm, NS2 protein has been shown to possess additional functions during the life cycle of influenza A virus including the contribution to viral budding and the accumulation of viral RNA resulting in the generation of RNA during the early stage of virus replication (Gorai et al., 2012; Mänz et al., 2012; Robb et al., 2009).
1.4.5 Nucleoprotein
NP of influenza A virus is encoded by segment 5. This protein is very conserved (about 95% sequence conservation at protein level) among influenza A viruses (Babar et al., 2015; Reid et al., 2004; Shu et al., 1993; Uyeki, 2003; Vemula et al.,2016; Yang et al., 2008), and abundant in virions. So NP has been used to develop methods for detection of influenza A viruses and for vaccine development against viral infection (Phuong et al., 2018; Zheng et al., 2014). NP protein includes two functional signals:
a nuclear localization signal (NLS) and a nuclear export signal (NES) (Bullido et al., Kudo et al., 1998; Wang et al., 1997; Weber et al., 1998; 2000; Yu et al., 2012) that play an important role in the transport of ribonucleoproteins (RNP), and interact with PB1 and PB2 directly (Biswas et al., 1998; Coloma et al., 2009; Medcalf et al., 1999;
Poole et al., 2004). NP is a multifunctional protein, which not only encapsidates the viral genome to form viral (vRNP) complexes together with PB1, PB2 and PA (Eisfeld et al., 2015), and modulates the replication and transcription of viral RNAs (Eisfeld et al., 2015; Jackson et al., 1982; Herz et al., 1981; Vreede et al., 2011), but also transports vRNP between nucleus and cytoplasm and helps to package the viral genome into new virions (Avalos et al., 1997; Digard et al., 1999; Martin and Helenius, 1991; O’Neill et al., 1995). In order to perform these functions, NP needs to interact with a wide range of factors of virus and infected cells (Eisfeld et al., 2015;
Wang et al., 2017; Watanabe et al., 2014). Furthermore, NP is also needed for the production of messenger RNA, viral genome RNA, and complementary positive-sense RNA in the viral life cycle.
1.4.6 Polymerase proteins and PB1-F2
Polymerase proteins of influenza A virus are crucial components in vRNPs, responsible for transcription and translation of viral RNA, and responsible for mutations in the viral genome during the viral life cycle for better adaptation to new hosts (Miotto et al., 2010; Neumann et al., 2004; Resa-Infante et al., 2011). The proteins include PA, PB1 and PB2 that are encoded by segment 3, segment 2, and segment 1, respectively. PA plays multiple functions in the viral life cycle including endonuclease activity of its N-terminal domain to cleave capped pre-mRNA of host origin, RNA promoter binding activity, and support for the polymerase subunits to form a functional complex (Dias et al., 2009; Hara et al., 2006; Kawaguchi et al., 2005; Maier et al., 2008; Sanz-Ezquerro et al., 1995; Yuan et al., 2009). PB1 contains domains which have the ability to bind the promoter of viral RNA and complimentary RNA within the vRNP and complimentary RNP, and play an important role in the RNA synthesis and in the assembly of the polymerase complex (González and Ortín J, 1999; González and Ortín J, 1999; Honda and Ishihama, 1997; Li et al., 1998).PB2 contains two separate sequences providing an RNA cap-binding site, which take caps from host pre-mRNAs in infected cells and initiate viral transcription (Fechter et al., 2003; Guilligay et al., 2008; Honda et al., 1999).
PB1-F2 is a second protein encoded by segment 2, and expressed from +1 within the open reading frame of PB1 gene (Chen et al., 2001). Almost all human isolates of
influenza A virus code for PB1-F2, many animals isolates do not encode this protein.
The full length PB1-F2 encoded by most isolates has 87 or 90 amino acids (Chen et al., 2001; Chen et al., 2004). The length of PB1-F2 in swine influenza viruses is different, for example, classical swine viruses code for a PB1-F2 with 8-11 amino acids, whereas Eurasian avian-like swine viruses code for a polypeptide of 87-89 amino acids (Neumann et al., 2009). This protein can induce apoptosis of infected cells via the by mitochondrial pathway, affect viral replication by interacting with PB1 protein, and affect the viral pathogenicity in mice (Chen et al., 2001; Conenello et al., 2007; Mazur et al., 2008; Zamarin et al., 2005).
2. Genetic variation of influenza A viruses
Mutations and reassortment events result in the generation of new strains and variants of influenza A viruses, which confer the ability to escape the pre-existing host immunity, to change pathogenicity and tissue tropism, and to overcome the species barrier to infect new hosts (Neumann et al., 2009). These new strains and variants are responsible for the yearly epidemics and occasional pandemics. That’s the reason why we have to update influenza vaccines to prevent the threat from influenza A viruses and reduce the economic losses though hosts infected by one subtype of influenza A virus can acquire lifelong immunity to the corresponding strain (Finkenstadt et al., 2005). Because of this antigenic variability and the ability to spread infection to susceptible hosts via air in a short time, influenza A viruses are listed as one of the most serious infectious pathogens that threaten the human population (Hu et al., 2017).
2.1 Point mutations
Mutations occur in the viral life cycle during the viral RNA replication in a different manner: substitutions (almost 1 × 10-3 to 8 × 10-3 sites/year) (Chen and Holmes, 2006), insertions and deletions, due to the viral polymerase which is responsible for replication and transcription of viral RNP but does not possess a proof-reading function (Aggarwal et al., 2010; Koelle et al., 2006; Nobusawa and Sato, 2006; Parvin et al., 1986; Smith et al., 2004). Mutations occurring in glycoproteins may change the viral antigenicity with the result that the pre-existing host immunity cannot prevent infection by these new strains. Several studies have shown that a single mutation occurring in an antigenic site of the H3 subtype may result in the change of antigenicity that enables mutant strains to evade the pre-existing immunity (Hensley et al., 2009; Horimoto and Kawaoka, 2001; Wiley et al., 1981). Mutations causing the characteristic changes of influenza A virus glycoproteins (HA and NA) are designated as antigen drift. In addition to antigenic changes, mutations may also cause a change in the pathogenicity and tropism of the virus. For example, the amino acid at position 627 of PB2 or 66 of PB1-F2 determine the pathogenicity of the H5N1 subtype in mice (Conenello et al., 2007; Hatta et al., 2001); with respect to the position 222 of the HA protein, amino acid D at this position confers the ability to preferentially grow in the
lung, whereas a G at this position enables to virus to grow efficiently in the trachea (Seidel et al., 2014). Furthermore, point mutations are crucial for the viruses in order to adapt to a new host; point mutations occur to survive in the new environment, and to make viral strains more stable (Castelán-Vega et al., 2014; Yang et al., 2017).
2.2 Reassortment events
The genome of influenza A viruses is composed of eight RNA segments, which provide the possibility of reassortment to generate new strains when different subtypes of influenza A viruses co-infect one host (Webster et al., 1982). In this way, an influenza virus may acquire gene segments from another virus, e.g. a human virus may acquire one or more genomic RNA segments from a swine virus. If a human virus acquires the genome segments coding for the viral glycoproteins HA and/or NA from an animal virus, the reassortant virus has antigenic properties that are completely different from those of the human parental virus. Such an event is designated as antigenic shift. Reassortment of influenza A viruses not only occurs in nature but also under laboratory conditions (Kreibich et al., 2013; Neumann et al., 2009). The events play an important role in the evolution of influenza A virus and may be associated with a change of pathogenicity and the switch of host (Dugan et al., 2008; Garten et al., 2009; Holmes et al., 2005; Scholtissek et al., 1978). In history, there were several pandemics in the human population that were caused by reassortant viruses. The most severe pandemic emerged in 1918 and was named Spanish flu. It was caused by an H1N1 virus that may have been transmitted directly from an avian host to humans (Horimoto and Kawaoka, 2005). The latest pandemic emerged in 2009. The genome of the viruses was derived from several subtypes of influenza A viruses. While the reassortment events of the triple reassortant precursor virus have occurred in swine, it is at present not known whether the final reassortment event has occurred in swine or humans (Neumann et al., 2009). Point mutations and reassortment events are the genetic basis for the generation of new virus strains and thus play an important role in the evolution of influenza viruses (Holmes et al., 2005; Nelson and Holmes, 2007).
3. Pigs and swine influenza A viruses 3.1 Pigs
Pigs are susceptible to infection by human and avian influenza A viruses. So genetic reassortment events can occur when a pig is infected by viruses of different subtypes and the two viruses infect the same cell in the airway epithelium (Scholtissek, 1990).
In this way, reassortant viruses have emerged in different parts of the world the genome of which is derived from human/swine influenza A viruses, human/avian influenza A viruses, or human/swine/avian influenza A viruses isolated from pigs in America and other countries (Olsen, 2002; Reperant et al, 2009). Therefore, pigs have been considered as “mixing vessel” for the generation of reassortant viruses (Ito et al., 1998; Ma et al., 2008). Because there is no pre-existing immunity against such new viruses, some of them can overcome species barriers to infect humans or animals of other species, and cause disease or pandemics. Therefore, pigs are considered to play an important role in the interspecies transmission (Horimoto and Kawaoka, 2001;
Imai and Kawaoka, 2012; Kida et al., 1994; Tanner et al., 2015). Previously, it has been tried to explain the role of pigs as mixing vessel for the generation of new influenza variants by the receptor distribution on the airway epithelium. It has been claimed that on the respiratory epithelium of pigs, there are both α-2,6-linked sialic acids and α-2,3-linked sialic acids, i.e receptor determinants for both human and avian influenza viruses. However, careful analysis has shown that the distribution of sialic acids in the human and porcine airways is very similar: α-2,6-linked sialic acids are most abundant in the upper airways; an increasing amount of α-2,3-linked sialic acids is detectable in the lower airways. Because of the similarity of the sialic acid distribution in humans and pigs, there have to be other factors to explain the role of pigs as “mixing vessels” (Nelli et al., 2010; Nicholls et al., 2007; Shinya et al., 2006).
3.2 Swine influenza A viruses
Swine influenza A viruses are common pathogens to infect airway epithelial cells in pig populations. The typical clinical symptoms of disease caused by swine influenza A viruses in pigs are loss of appetite, fever, depression, labored breathing, and coughing which can result in poor growth, weight loss and economic losses. The disease in pigs is characterized by high morbidity and low mortality (Kyriakis et al.,
2013). Swine influenza was first described in pigs in America in 1918 when the Spanish flu pandemic occurred (Taubenberger et al., 2001). It was not until 1930, that the first swine influenza A virus (classical swine influenza virus H1N1) was isolated from a pig (Shope, 1931). Although subtypes H1N1, H3N2 and H1N2 of swine influenza A viruses are circulating in pigs worldwide, these viruses are different in their genetic properties and origin on the basis of their geographic distribution (Kuntz-Simon and Madec, 2009; Kyriakis et al., 2013).
3.2.1 Swine influenza A viruses in North America
In North America, the classical swine H1N1 virus lineage had been present and stable in pig populations since this virus was first detected in 1918 until 1998 (Vincent et al., 2014). After 1998, reassortant H3N2 viruses emerged with RNA segments encoding HA, NA and PB1 derived from human influenza A virus of the H3N2 subtype, with segments encoding M, NP and NS derived from classical swine influenza virus (H1N1), and the rest, i.e. PB2 and PA being derived from avian influenza A virus.
This reassortant virus established a new lineage in pig population in America (Webby et al., 2000; Zhou et al., 1999). From that time on, reassortant virus H3N2 and classical swine influenza A virus H1N1 were co-circulating in swine herds and generated another two subtypes, H1N1 and H1N2 (Karasin et al., 2002; Webby et al., 2004). In 2005, human-like H1N1 and H1N2 strains with HA or/and NA acquired from human influenza A viruses were present in pig populations in the United States (Vincent et al., 2009). In 2009, a newly reassortant H1N1 virus emerged and caused a pandemic in the human population. This virus is also present in American pig populations
3.2.2 Swine influenza A viruses in Europe
In Europe, classical swine influenza A virus (H1N1) was prevalent in swine herds until 1979 and replaced by a fully avian-origin H1N1, which entered pig populations and was designated as avian-like swine influenza A virus (Dunham et al., 2009;
Pensaert et al., 1981). In the 1980s, a new H3N2 virus spread in pigs in Europe, which was generated by a reassortment event between human A/Hong Kong/1/68 (H3N2) virus and avian-like swine influenza A virus H1N1. The former provided the RNA segments of HA and NA, the latter provided the rest of the RNA segments (Castrucci
et al., 1993; de Jong er al., 1999; Haesebrouck et al., 1985). Sometime later, another reassortant virus, H1N2, emerged in pigs in Western Europe. This virus maintained the genome of the reassortant H3N2 virus except for the HA gene which was derived from a human H1N1 virus (Brown et al., 1998; Kyriakis et al., 2011; Marozin et al., 2002; Schrader and Süss, 2003). The results of surveillance studies in pigs within Europe since 2010 show that after the 2009 pandemic, Eurasian avian-like H1N1, human-like H3N2 and human-like H1N2 viruses continued to be prevalent in swine populations, but in addition also swine-origin pandemic H1N1viruses are present now (Vincent et al., 2014).
4. Pandemics
Historically, there were several pandemics caused by influenza A viruses including the Spanish influenza in 1918, the Asian influenza in 1957, the Hong Kong influenza in 1968, the Russian influenza in 1977 and the swine-origin influenza in 2009 (Fig. 3).
Pandemics were caused by reassortant viruses the genome of which is derived from different subtypes of influenza A viruses. These reassortant viruses possess an HA protein which is derived from avian influenza viruses (Horimoto and Kawaoka, 2005).
Fig. 3 Pandemic events of influenza A virus (Wendel et al., 2015) 4.1 Spanish influenza pandemic in 1918
The Spanish influenza pandemic was caused by an avian-like influenza A virus H1N1, which also possessed some human-like amino acid residues in several proteins based on the analysis of genomic sequences (Rabadan et al., 2006; Taubenberger et al., 2005). As genomic sequences of influenza A viruses from the time before 1918 are
not available, the parental viruses of this avian-like virus are unknown (Smith et al., 2009). In 1918-1919, around 50 million people in the world were killed by this pandemic which is the most devastating epidemic known in history (Johnson and Mueller, 2002). The high mortality was due to bacterial pneumonia by co-infecting bacteria. As no antibiotics were available at that time, there were no therapeutic countermeasures possible (Morens et al., 2008).
4.2 The Asian influenza pandemic in 1957
The Asian influenza pandemic emerged in 1957 and was caused by a human/avian reassortant H2N2 virus with the genomic segments of HA, NA and PB1 derived from avian H2N2 subtype and the rest of the segments derived from the lineage of the 1918 pandemic virus (Kawaoka et al., 1989; Scholtissek et al., 1978). This pandemic originated in the South of China and then spread to Japan, United States and Britain.
About 70,000 people were killed in this pandemic (Neumann et al., 2009).
4.3 The Hong Kong influenza pandemic in 1968
In 1968, the Asian pandemic virus H2N2 was replaced by another human/avian reassortant virus, H3N2, which caused the Hong Kong pandemic. The gene segments encoding for HA and PB1 proteins were acquired from an H3 subtype of avian influenza A viruses, the other six gene segments derived from the Asian pandemic virus H2N2 (Kawaoka et al., 1989; Scholtissek et al., 1978). This pandemic emerged in the winter of 1968 and 1969, and resulted in the death of around 33,800 people in the United States (Neumann et al., 2009).
4.4 The Russian influenza pandemicn in 1977
The Russian pandemic emerged in 1977 and was caused by H1N1 virus which was very similar to the H1N1 virus circulating in the early 1950s on the basis of genetic analysis (Nakajima et al., 1978). This H1N1 virus was prevalent in the Northern hemisphere in 1977-1987, and didn’t replace the Hong Kong pandemic virus H3N2;
since then viruses of both subtypes are co-circulating in the human population (Neumann et al., 2009; Taubenberger and Kash, 2010).
4.5 The Swine-origin influenza pandemic in 2009
The latest pandemic emerged in 2009 in Mexico, and was caused by a novel H1N1
virus which was generated by reassortment events between two swine influenza A viruses: Eurasian avian-like swine virus H1N1 and triple reassortant swine virus H1N2 (Garten et al., 2009; Neumann et al., 2009). Data of genetic sequences show that the gene segments of NA and M are acquired from Eurasian avian-like swine virus H1N1 lineage, gene segments of HA, NP and NS are derived from classical swine virus H1N1, gene segments of PB2 and PA are derived from an avian virus H1N1, and gene segment of PB1 is derived from a human seasonal virus H3N2 (Smith et al., 2009; Wendel et al., 2015). This novel H1N1 virus was transmitted from pigs to humans and killed around 150 000 to 570 000 people worldwide during the first 12 months since its emergence (Garten et al., 2009; WHO 2010; Dawood et al., 2012). Currently, the H1N1 virus is circulating in humans and pigs with point mutations to adapt to the new host and to become stable for survival in the new environment (Castelán-Vega et al., 2014).
5. Barrier function of the respiratory tract
Airway epithelial cells, classified into ciliated cells, goblet cells and basal cells (Fig.4), line the respiratory tract and are target cells for the infection by different respiratory pathogens. The surface of the airway epithelium has contact with the external environment. Due to the large surface area of the respiratory tract (a surface area of 2500 cm2 covered by around 1010 cells in humans) and the large quantity of air taken up per minute (6-12 L/min in humans), the respiratory epithelium is continuously exposed to viruses, bacteria and physical particles present in the inhaled air (Ganesan et al., 2013; Mercer et al., 1994). In order to prevent the detrimental effect of foreign substances, the respiratory tract is equipped with a barrier function: mucociliary clearance system, intercellular junction complexes and antimicrobial peptides (Fig.4) (Ganesan et al., 2013; Tilley et al., 2015).
Fig.4 Structure of epithelial cells of respiratory tract (Ganesan et al., 2013) 5.1 Mucociliary clearance system
The mucociliary clearance system is the first line of defence against external invading agents, its function is based on specialized airway epithelial cells: ciliated cells and goblet cells. Goblet cells release mucins which are high molecular weight glycoproteins to form a mucus layer that can entrap foreign substances including viruses, bacteria and physical particles (Rubin, 2002). The removal of entrapped foreign substances out of the respiratory tract is accomplished by ciliated cells via their ciliary activity. Each ciliated cell possesses around 200 to 300 cilia the length of which is different in different parts of the respiratory tract (Serafini and Michaelson,
1977). The frequency of ciliary beating and the hydration of mucus determine the transport efficiency (Puchelle et al., 1995; Tarran et al., 2001). Mitochondria beneath the apical surface of ciliated cells provide the energy for ciliary beating (Harkema et al., New york: Marcel Dekker, 1991). The cilia are surrounded by a fluid of low viscosity (compared to mucus layer) which forms the periciliary layer and modulates the coordinated movement of cilia (Knowles and Boucher, 2002; Randell et al., 2006;
Tarran, 2004). The mucus layer is floating on top of the periciliary layer; together they function as mucociliary defence mechanism to protect the respiratory tract.
5.2 Intercellular junction complexes
Intercellular junction complexes, tight junctions and adherens junctions, connect the cells of the airway epithelium tightly and thus contribute to the barrier function. Tight junctions contain transmembrane proteins including occludin, claudins and junctional adhesion molecule that are responsible for membrane connection from cell to cell, and scaffolding proteins including ZO-1, ZO-2 and ZO-3 that help the transmembrane proteins to locate at the cytoskeleton. (Ganesan et al., 2013; Hartsock and Nelson, 2008; Schneeberger and Lynch, 2004; Shin et al., 2006). Adherens junctions mainly contain the transmembrane glycoproteins cadherin and catenin that play a role in the connection of adjacent cells (Ganesan et al., 2013; Hartsock and Nelson, 2008). In addition, these two junctions modulate the paracellular permeability of epithelial cells of the respiratory tract (Pohl et al., 2009). Under healthy conditions, intercellular junction complexes protect the airway epithelium from harmful effects by respiratory pathogens and other particles, and they play a role in the proliferation and differentiation of cells (Balda and Matter, 2009; Koch and Nusrat, 2009).
5.3 Antimicrobial peptides
In addition to the physical barrier made up by the mucociliary clearance system and intercellular junction complexes, the respiratory tract also contains a biochemical barrier formed by lots of different antimicrobial material released by epithelial cells (Ganesan et al., 2013). The antimicrobial material is present in the airway surface liquid and acts against pathogens intruding via inhaled air. For example, lysozyme, one of the enzymes released by airway epithelial cells, acts not only efficiently against gram-positive bacteria, but also gram-negative bacteria together with lactoferrin
(Ellison and Giehl, 1991; Ibrahim et al., 2002); Human β-defensins, one cluster of antimicrobial peptides expressed in the airway surface liquid, can efficiently fight against a wide range of pathogens including viruses and bacteria (Ganz, 2003; Kota et al., 2008; McCray and Bentley, 1997).
6. Primary culture systems of differentiated airway epithelial cells
As the respiratory tract is exposed continuously to the external environment, airway epithelial cells are important target cells for different respiratory pathogens, and therefore at an increased risk of infection by viruses and bacteria. In order to prevent detrimental effects on the respiratory tract, airway epithelial cells possess barrier functions, such as mucociliary clearance system which is the first line of defence against harm from outside. The full set of functional barriers is not present in immortalized cell lines. While some immortalized cell lines, e.g. MDCK contain tight junctions, other functions are only present on differentiated airway epithelial cells.
The latter cells contain ciliated cells, mucus-producing cells, basal cells and other cells that are characteristic for the respiratory tract and thus provide a suitable model to imitate the natural situation in the host. Primary culture systems of differentiated airway epithelial cells provide a suitable tool to analyze the interaction of respiratory pathogens and epithelial cells under natural conditions. Several primary culture systems have been established from different species (Bearson et al., 2003; Kirchhoff et al., 2014; Meng et al., 2013; Meng et al., 2016; Wu et al., 2016; Yachida et al., 1978). Here I will introduce two of them: precision-cut lung slices (PCLS) and air-liquid interface (ALI) cultures.
6.1 Precision-cut lung slices
Since Martin et al had prepared slices from rats with a thickness of 250µm + 20 in 1996 (Martin et al., 1996), the culture system of precision-cut lung slices (PCLS) has been established from different animal species including bovine, avian, porcine, and ovine airways (Abd EI Rahman et al., 2010; Goris et al., 2009; Lambermont et al., 2014; Meng et al., 2013). This culture system provides an ex vivo model for airway epithelial cells which retains the original setting of cells as found in the host. The advantages of this culture system are: large numbers of slices can be generated from one lung; ciliated cells, mucus-producing cells and basal cells are present in each slice;
the viability of slices can be maintained for more than 7 days under ex vivo conditions (Meng et al., 2013; Punyadarsaniya et al., 2011). This interesting culture system has been used for different purposes of study, such as to evaluate the growth property of different subtypes of swine influenza A virus. The ciliary activity can serve as an
indicator for the viral virulence (Meng et al., 2013). PCLS have been used to investigate the infection strategy and the target cell tropism of three viruses which are associated with the bovine respiratory disease complex (Goris et al., 2009; Kirchhoff et al., 2014). Furthermore, they have been applied to analyze the adaptation of an H9N2 avian influenza A virus to swine cells (Yang et al., 2017). PCLS were also applied for co-infection experiments to analyze the interaction between swine influenza A viruses and Streptococcus suis (Meng et al., 2013).
6.2 Air-liquid interface cultures
Air-liquid interface cultures provide an in vitro model for differentiated airway epithelial cells which can be used to analyze the infection by respiratory pathogens.
The primary cells derived from trachea or bronchi can be induced to differentiate into ciliated cells, mucus-producing cells, basal cells and other cells under air-liquid interface conditions (Fulcher et al., 2015; Meng et al., 2016; Wu et al., 2016). Up to now, this culture system has been developed for different species including human, swine, bovine, ferrets and murine (Fulcher et al., 2015; Kirchhoff et al.,2014; Liu et al., 2007; Meng et al., 2016; Wu et al., 2016). ALI cultures facilitate studies about the effect of respiratory pathogens on the respiratory barrier functions: mucociliary clearance system and intercellular junction complexes (Wu et al., 2016), and provide a platform to characterize pathogens which do not efficiently infect immortalized cell lines and animal models, like human coronaviruses (Dijkman et al., 2013; S Banach et al., 2009). ALI cultures have been used to analyze co-infection (Nguyen et al., 2015) and the characteristics of respiratory pathogens, such as coronavirus, influenza A viruses, parainfluenza viruses, respiratory syncytial virus, and Streptococcus
(Dijkman et al., 2013; Goris et al., 2009; Meng et al., 2016; S Banach et al., 2009; Wu et al., 2016).
Taken together, precision-cut lung slices and air-liquid interface cultures of differentiated airway epithelial cells are promising tools to study the properties of respiratory pathogens, and the interaction of specialized cells with pathogens.
7. Infection of pigs by five A(H1N1)pdm09 viruses
Five strains of influenza viruses that were derived from the pandemic virus of 2009 have been isolated in the years between 2009 and 2015 (HA09, JE09, JE10, SC14, and KI15). These viruses have been recently applied by our collaboration partner Ralf Dürrwald in pig experiments to compare them with respect to their virulence. As the results are crucial for the interpretation of my in vitro-experiments, they are described here shortly.
7.1 Dyspnea and rectal temperature of animals infected with A(H1N1)pdm09 viruses
Pigs were infected with the five viruses by using high-dose aerosol nebulisation. At the indicated times, dyspnea and rectal temperature of clinical symptoms caused by the viruses were determined. As shown in Fig. 5, HA09, JE09 and JE10 strains from 2009/2010 caused severe dyspnea in infected pigs, but SC14 and KI15 isolates from 2014/2015 did not induce these symptoms. Among the three strains from 2009/2010, the HA09 isolate induced the most severe dyspnea in infected pigs compared to JE09 and JE10 strains (Fig. 5). HA09 was also the only strain that caused the death in some infected pigs within 2-6 days post-infection with a mortality of 18%; JE09 and JE10 strains induced the strongest dyspnea at 1 day post-infection (Fig. 5); apart from 1 day post infection, another dyspnea peak was observed after infection by JE09 strain from 4 to 6 days post infection (Fig. 5). SC14 and KI15 strains only induced a slight increase at the early stage of the infection period (Fig. 5).
Regarding the rectal temperature, the viruses of 2009/2010 induced fever (higher than 40oC) at 1 day post infection, while the isolates of SC14 and KI15 failed to do so and did not affect the rectal temperature in infected pigs (Fig. 6).
Fig. 5 Dyspnea in pigs infected with the five viruses
Dyspnea score (arithmetic mean) in infected pigs with the five isolates of 2009/2010/2014/2015 was investigated at indicated times; dpi, days after infection; m, morning; a, afternoon. There were no significant differences between viruses SC14 and KI15; therefore all statistical calculations were done with the viruses isolated in 2009/2010 (HA09, JE09, JE10) in comparison to KI15.
Fig. 6 Rectal temperatures in pigs infected with the five viruses
Rectal temperatures (°C, arithmetic mean) were measured after infection in pigs by the five isolates of 2009/2010/2014/2015 at indicated times; dpi, days after infection;
m, morning; a, afternoon. There were no significant differences between viruses SC14 and KI15; therefore all statistical calculations were done with the viruses isolated in 2009/2010 (HA09, JE09, JE10) in comparison to KI15.
7.2 Lung lesions and viral load in the lung of pigs infected with A(H1N1)pdm09 viruses
Apart from dyspnea score and rectal temperature, two more parameters were determined: lung lesions and viral load in the lung. As shown in Fig.7, lung lesions induced by 2009/2010 isolates were more pronounced than lesions induced by viruses of from 2014/2015, and they were detectable up to nine days post infection. Strains of 2014/2015 only induced mild lung lesions in infected pigs (Fig. 7).
In all pigs infected by either of the five influenza strains, virus was detectable in the lungs at 1 day and 3 days post-infection, but not at 9 days post-infection. As shown in Fig. 8, the viral load in the lungs of pigs infected by viruses from 2009/2010 was significantly higher than the load of virus in animals infected by 2014/2015 viruses.
Among viruses from 2009/2010, the viral load in the lungs induced by HA09 and JE09 strains at 1 day post infection was more pronounced compared to the isolates from 2010 (Fig. 8); at 3 days post infection, HA09 strain induced a significantly higher virus load in lungs compared to JE09 and JE10 isolates (Fig. 8).
Taken together, the animal experiments indicate that the pathogenicity/virulence of the viruses from 2014/2015 for pigs was significantly decreased compared to the viruses from 2009/2010; the swine-derived virus SC14 strain and the human-derived virus KI15 strain shared the properties in pig infections, i.e. there was no difference between these two strains as far as their virulence is concerned.
Fig. 7 Lung lesions in pigs infected with the five viruses
Gross lung lesions (%, arithmetic mean with standard deviation) were analyzed after infection in pigs by the five isolates of 2009/2010/2014/2015 at indicated times; dpi, days after infection; m, morning; a, afternoon. There were no significant differences between viruses SC14 and KI15; therefore all statistical calculations were done with viruses HA09, JE09, JE10 in comparison to KI15; 3.5 = detection limit due to predilutions of the samples.
Fig. 8 Viral lung load in pigs infected with the five viruses
The viral load in lungs (lg TCID50, geometric mean with standard deviation), was determined after infection in pigs by the five isolates of 2009/2010/2014/2015 at indicated times; dpi, days after infection; m, morning; a, afternoon. There were no significant differences between viruses SC14 and KI15; therefore all statistical calculations were done with viruses HA09, JE09, JE10 in comparison to KI15; 3.5 = detection limit due to pre-dilutions of the samples.
8. Aim of the study
Aim of this study is to analyze the interaction of influenza A viruses and airway epithelial cells by using influenza A viruses and two primary cell culture systems:
porcine precision-cut lung slices and air-liquid interface culture.
The first defence line of the respiratory tract is the mucociliary clearance system derived from specialized cells: mucus-producing cells and ciliated cells, the former release mucins which can entrap foreign material and the latter transport mucus out of the respiratory tract via ciliary beating to protect the host. We were interested whether cilia possess a role in impeding virus infection in addition to their transport function.
The culture system of interest is porcine PCLS, as in this ex vivo model the ciliary beating can be observed conveniently under the light microscope. As a respiratory pathogen we chose the H3N2 subtype of swine influenza A viruses, as pigs are an important host for influenza A viruses, and the H3N2 virus is one of the subtypes circulating in pig populations.
Swine-origin influenza A virus of the H1N1 subtype emerged in 2009 and was transmitted from pigs to humans causing a pandemic. To evaluate whether the virulence of strains from human and swine isolated at the beginning year and the years following 2009 changed, porcine air-liquid interface cultures were chosen as an in vitro model to be infected by these strains. ALI culture systems of differentiated airway epithelial cells possess some parameters derived from mucociliary clearance system and intercellular junction complexes that can be used to reflect viral effects in the airway epithelium. In addition, amino acid sequences of proteins from different isolates are available and can be compared. The data of my in vitro-experiments, should be compared to animal experiments that have been performed by our collaboration partner Ralf Dürrwald (summarized in Introduction, section 7). All these data together should provide a better understanding of the viral virulence.