Final Report Summary - FLUPIG (Pathogenesis and transmission of influenza virus in pigs)
Executive Summary:
The FLUPIG project aimed to gain better insights into the role of pigs in the generation of novel pandemic influenza viruses for humans. We have performed in vivo infection studies with avian H1N1 and H9N2 influenza viruses in pigs, ferrets and birds with the purpose to identify factors that mediate adaptation of avian influenza viruses to pigs (WP 1), as well as transmission of these viruses between pigs (WP 2) and from pigs to other animal species (WP 3). All three steps would be required to turn a wholly avian virus into a pandemic virus. The process of adaptation of avian influenza viruses to pigs (WP 1) was studied both at the level of the host and virus. The swine host was found to be similar to humans in some aspects, but not in other. For example, the prevalence and distribution of “avian-type” and “human-type” influenza virus receptors in the airways appeared to be almost identical in pigs and in humans. The avian-type receptor was very scarce in the nasal mucosa and trachea and largely restricted to the lungs, whereas the human-type receptor was found throughout the respiratory tract. However, there appeared to be differences between pigs and humans in the stage of virus replication that follows attachment of the virus to host receptors. Indeed, the pH optimum at which the viral surface protein hemagglutinin (HA) undergoes a conformational change that triggers the so-called fusion of the virus with the host cell was found to be higher for swine than for human influenza viruses. We used two different approaches to try to adapt avian influenza viruses to pigs: a) blind serial passages with wholly avian influenza viruses in the pig, with the purpose to introduce mutations that make the virus more fit to replicate in swine and b) genetic “re-assortment” in the lab, resulting in a hybrid virus with surface protein genes of the avian H9N2 virus and internal protein genes of the 2009 pandemic H1N1 virus. The latter virus is known to be well adapted for replication in both swine and humans. Both approaches resulted in a slight to moderate enhancement of avian influenza virus replication in the pig, but they were insufficient to make the avian viruses fully swine adapted. Though the pig-passaged or reassortant viruses were able to transmit between pigs, transmission remained highly inefficient when compared to that of natural swine adapted viruses. Genetic analyses of pig-passaged viruses revealed several mutations in the HA as well as other viral genes. Remarkably, replication efficiency and transmission of the wholly avian H1N1 influenza virus was much better in ferrets than in pigs, which suggests that the pig and ferret present different species barriers. The question as to whether viruses with an H9, which have not become established in the swine or human population so far, may ever become adapted to pigs as H1 and H3 did remained unanswered. While it is possible that a different or more forced approach would result in fully swine adapted viruses, adaptation of avian viruses to the pig clearly does not occur easily. Interestingly, adaptation of a duck H1N1 virus to pigs made the virus less fit for replication in birds. Pandemic influenza viruses must not only have the ability to spread efficiently from person to person, another requirement is that immunity within the human population must be sufficiently low to allow widespread infection. Studies in pigs and/or mice showed substantial cross-protection between antigenically distinct influenza viruses within a given HA subtype, but only limited cross-protection between viruses with a different HA subtype (WP 4). The consortium also developed novel live vaccines for swine influenza virus that were stably attenuated by biotechnology. Both vaccines gave promising results in challenge studies with the homologous virus included in the vaccine and their ability to protect against more diverse viruses should be examined in the future.
Project Context and Objectives:
Influenza A viruses originate in wild birds and are transmitted to other avian and mammalian species (including humans) in which they may cause self-limiting infections, epidemics, or panzootics. A few times each century they cause pandemics. The mechanisms by which influenza viruses gain the capacity of abandoning the animal reservoir and becoming widespread in human beings are currently unknown.
Among all animals, the pig is believed to play an essential role in the influenza virus ecology, since it has been shown that (1) pigs are susceptible to all subtypes of influenza A viruses, including those of avian origin; (2) pigs have receptors for both avian and mammalian origin viruses, and thus represent an ideal vessel for viral reassortment or adaptation of an avian virus to the mammalian host. In fact, influenza viruses isolated from swine often contain genes, which belong to viruses isolated from other species. The most recent example is the 2009 H1N1 pandemic virus, which has originated from swine herds in North America. It is a reassortant, which contains genes which can be traced back to three species (avian, human and swine) and two hemispheres. Six gene segments are similar to ones previously found in triple reassortant swine influenza viruses (SIVs) circulating in pigs in North America, from which the pandemic virus has derived a classical H1 haemagglutinin (HA). Of these 6 genes, three (HA, nucleoprotein (NP), non-structural protein (NS)) had been established in viruses endemic in the pig population for decades, Polymerases PB2 and PA were introduced into pigs from the avian host in 1998 and the PB1 appears to be derived from a human virus introduced in 1998. The two other gene segments, encoding the N1 neuraminidase (NA) and matrix proteins (MP) are derived from a virus circulating in swine populations in Europe or Asia. Thus, this virus has a core of genes that had been circulating in SIVs for over 30 years, which has acquired in more recent times two avian genes and one human gene.
It should be stressed however, that there is no direct evidence that pigs have played a role in the genesis of any of the three pandemics of the 20th century. In other words, the hypothesis of the pig as an intermediate host between birds and humans has never been proven for any of the pandemic viruses of the last century. In fact, the swine-human link for both the 1957 and 1968 pandemic viruses (both of which were reassortants between the human virus circulating at the time and an avian virus of a novel HA subtype) has not been established.
Both in the current and last century there have also been documented episodes of transmission of influenza virus from swine to humans, although these SIVs failed to spread further in the human population. The reason for this is unclear. In addition, as far as we know, most SIVs of the H1N1, H3N2 and H1N2 subtypes are endemic in the swine population and only sporadically cause human infection.
Though the pandemic (H1N1) 2009 influenza virus almost certainly comes from pigs, it is still unclear why and how this novel H1N1 virus obtained the capacity for human-to-human spread, while the established SIVs have so far failed to do so. On the other hand, we have evidence that all known pandemic viruses contain an avian genetic component – and it is unclear what role is played by genes of avian origin in the generation of pandemic viruses, particularly in view of the widespread infection of poultry with H5N1 and H9N2 viruses. Co-circulation of the pandemic (H1N1) 2009 influenza virus and other animal influenza viruses (avian H5N1 and H9N2 and swine H3N2, H1N1 and H1N2) in susceptible hosts could represent the occasion for viral reassortment with unpredictable consequences.
Another aspect that is to be considered is that pigs support the endemic circulation of viruses that belong to the same subtype of viruses that have been shown (to date) to cause pandemics in humans. Understanding how the immune system of pigs responds to infection and the extent of cross-protection within and between virus subtypes is crucial to limiting the degree of circulation of these viruses in pigs, as is the development of novel vaccination strategies.
Though obviously a rare event, there is a real danger that swine-origin reassortants start to spread readily between humans and launch a pandemic, and this is perfectly illustrated by the novel H1N1 virus. In order to proactively respond to such threats in the future, we will need to fill in the lack of knowledge about the role of pigs in overall influenza ecology. In order to do so, following questions have to be addressed:
Q1. What are the mechanisms whereby an avian virus is able to adapt in the pig host and subsequently establish a new lineage in pigs, and what is the true significance of pigs as a mixing vessel?
Q2. What determines the transmissibility of influenza virus between pigs?
Q3. What determines the transmissibility of influenza virus from pigs to other relevant animal species, including ferrets as a model for humans?
Q4. What determines the transmissibility of influenza virus from ferret to ferret as a model for human-to-human transmission?
Q5. Can previous infections or vaccinations with influenza virus protect against novel, antigenically different viruses?
The FLUPIG consortium aimed to study each of these questions based on its solid experience in influenza pathogenesis and transmission in a wide variety of ex vivo models as well as in vivo animal models, including swine. In particular, we aimed at improving our knowledge on the gene constellation and genetic interactions that are necessary to generate pandemic viruses in combination with an improved understanding of the host-dependent variables such as receptor distribution and immune response. Gaining insight in the characteristics of the pathogen and combining this with the host component will be the main goal of our consortium. Combined with improved surveillance for influenza in animals, effective vaccines and antiviral drugs, this knowledge will be critical to the control of future influenza pandemics.
The specific objectives of the consortium were:
Study the replication efficiency of different viruses in pigs in vivo, as well as in reliable in vitro models
− Identify specific viral amino acid signatures and gene constellations that enhance the replication of influenza viruses in pigs;
− Determine whether viruses with an H9, which have not become established in the swine or human population so far, may ever become adapted in pigs as H1 and H3 did;
− Gain insights into the host factors, including virus receptors, that contribute to the replication efficiency of influenza viruses in pigs.
Study the transmissibility of different viruses between pigs
− Investigate mechanisms whereby non-swine HA and NA restrict virus transmissibility in pigs;
− Determine the genetic factors that influence virus transmissibility and pathogenesis of the pandemic (H1N1) 2009 influenza virusin pigs;
− Identify genetic changes in influenza viruses necessary for acquisition of swine and human transmissibility;
− Compare virus transmissibility in the swine model with that of ferrets and in vitro/ex vivo tools.
Study the transmissibility of different viruses from pigs to other relevant animal species
− Investigate and compare the capabilities of SIVs to be transmitted from pigs to other relevant animal species, with particular reference to birds;
− Investigate and compare the capabilities of SIVs to be transmitted from pigs to ferrets, as a representative model for humans;
− Determine the role of selected swine influenza virus (SIV) genetic determinants for inter-species transmission.
To study heterovariant and heterosubtypic cross-protection between influenza viruses in pigs
− To identify the level of cross-protective immunity between H1N1 viruses with an antigenically different H1 (heterovariant protection) (e.g. European swine H1N1 virus versus novel 2009 H1N1 pandemic virus; human seasonal H1N1 viruses versus novel 2009 H1N1 pandemic virus);
− To identify the level of cross-protective immunity between SIVs belonging to different HA subtypes or between SIVs and avian influenza viruses (AIVs) (heterosubtypic protection);
− To identify the immune mechanisms mediating cross-protection between influenza viruses;
− To evaluate novel modified-live attenuated vaccine (MLV) candidates against swine influenza.
Project Results:
Scientific and technical results and foreground
For a long period of time, there is the hypothesis that pigs play a key role in the development of influenza viruses with pandemic potential. Several arguments were raised to support that hypothesis, but review of historical data indicated that the hypothesis is an oversimplification. Therefore, the FLUPIG consortium aimed at gaining more insight in the role of the pig by addressing several of the traditional assumptions in experimental studies. An overview of the results obtained and their relevance in view of the role of the pig are briefly outlined below.
The susceptibility of pigs to influenza viruses of avian origin: the species barrier
Historical data point out that direct avian-to-pig transmission of influenza viruses regularly occurred under natural conditions (Pensaert et al. 1981; Peiris et al. 2001; Guan et al. 1996; Loeffen et al. 2004). Furthermore, experiments performed by Kida and colleagues (Kida et al. 1994) proved that pigs are receptive to a wide variety of AIVs, whereas an earlier experimental study in human volunteers showed that humans were largely refractory to infection with certain AIVs (Beare and Webster 1991). However, while the pigs in the study of Kida et al. were fully influenza-naïve, most human volunteers had previously been infected with influenza viruses, so it cannot be excluded that this affected their susceptibility to AIVs.
Another argument that the unique susceptibility of pigs to AIVs is not as obvious as previously believed is provided by the recent outbreaks with a highly pathogenic avian H5N1, with an avian influenza H7N7 virus and with an avian H7N9 virus. The H5N1 outbreak is the largest and most devastating outbreak of highly pathogenic avian influenza in history, not only affecting millions of chickens and ducks, but also having a serious impact on human health with an exceptionally high case-fatality rate. However, most, if not all, of the human cases were in people with close contact with infected poultry and direct exposure to large amounts of H5N1 virus. Pigs can and do become infected with avian H5 subtypes under natural and experimental conditions, but all evidence indicates that the currently circulating H5N1 viruses are not well adapted for replication in pigs (Lipatov et al. 2008; De Vleeschauwer et al. 2009; De Vleeschauwer and Van Reeth 2010). The few available experimental infection studies indicate that the H5N1 virus replicates in the respiratory tract of pigs, but to a much lower extent than typical SIVs and mainly in the lungs. Remarkably, the pigs showed only mild or no clinical signs, while the viruses were extremely virulent for poultry. Also, the infected pigs failed to transmit the virus to uninfected littermates.
Key FLUPIG data in view of the species barrier hypothesis:
- Wholly avian H1N1 (duck/Bavaria/77) and H9N2 (quail/Hong kong/G1/97) viruses replicated in pigs, but replication was highly inefficient when compared to that of typical swine-adapted influenza viruses. Though there was limited pig-to-pig transmission of these viruses under experimental conditions, transmission as well was highly inefficient when compared to that of typical SIVs.
- Serial (up to 25), blind passages with these avian H1N1 and H9N2 viruses resulted in a slightly enhanced replication and/or transmission efficiency, but we failed to obtain fully swine adapted viruses by this adaptation. In addition, the “partially swine-adapted” H9N2 virus was lost again upon further passages.
- The pig passaged avian H1N1 viruses showed several mutations that are similar to those observed in early isolates of the “avian-like” H1N1 SIV, including mutations in the receptor-binding site that are known to enhance binding to the “human-type” receptors. However, none of the viruses acquired a full set of mutations found in natural swine viruses.
- The unadapted wholly avian H1N1 virus showed a better replication in and transmission between ferrets than in pigs. This suggests that the pig and the ferret present different barriers for HA and NA functions.
- Similarly a reverse-genetics engineered H9N2 reassortant virus, with the avian HA and NA in a backbone of a 2009 pandemic H1N1 virus, showed a slightly better replication in and transmission between pigs than the wholly avian H9N2 virus. But this approach, as well pig-passage with the reassortant virus, were also insufficient to make the avian virus fully adapted to pigs.
- In in vitro viral attachment assays, an avian H7N9 virus showed only limited attachment to the nasal mucosa of pigs. Stronger attachment was observed with nasal mucosa of human, ferrets, macaques and guinea pigs.
- Our data underscore the difficulty of reproducing virus adaptation in an experimental setting. It is possible that adaptation would have occurred with a more forced approach or with a combination of lab-made genetic changes to the viruses (e.g. reassortment in addition to specific viral mutations) and passages.
The pattern of receptor expression needed to enter into the respiratory tract
Another reason why pigs were appointed as prime candidates for the role as intermediate hosts is related to the nature of the influenza receptors in the respiratory tract. Binding of a virus to its receptor is the very first step in the viral replication cycle. For an influenza infection, binding is accomplished between the viral HA protein and the cellular sialic acid site. Although all influenza viruses recognize sialic acid, the type of sialic acid linkage with its underlying sugar residue as well as the receptor specificity of the viral HA differ between host species. AIVs preferentially bind to the sialic acid α 2,3 Gal linkage type (2-3 Sia), whereas human influenza viruses favour binding to the sialic acid α 2,6 Gal linkage type (2-6 Sia). Subsequently, it was found that the preferential replication site for influenza in ducks expressed α 2,3 Gal (Ito et al. 1998), whereas the human respiratory tract mainly expressed the α 2,6 Gal (Baum and Paulson 1990). As the pig trachea was found to contain both receptors, it was easily concluded that the pig could allow (1) adaptation of the virus from avian-type to human-type receptors providing a potential link from birds to humans (Ito et al. 1998; Ito 2000) and/or (2) simultaneous infection of a host cell with an avian and human virus and subsequent genetic reassortment.
More recent data showed that the sialic acid expression in the various host species is not as straightforward as initially thought. In fact, Van Poucke et al. (Van Poucke et al. 2010) could not confirm the studies of Ito et al., (1998). They found moderate to abundant expression of the human-like virus receptors in nasal, tracheal, bronchial and lung explants of pigs, but only a minimal expression of the avian-like receptors, especially in the nasal, tracheal and bronchial epithelium. The lack of α 2,3 Gal receptor expression correlated well with a lack of avian virus replication. The ex vivo studies of Van Poucke and colleagues are in agreement with the virus-binding experiments of van Riel et al. (van Riel et al. 2007). Additional studies of the human respiratory tract demonstrated a receptor distribution pattern similar to that in pigs with the presence of both sialic acid types (Shinya et al. 2006). Matrosovich et al. (Matrosovich et al. 2004) and Thompson et al. (Thompson et al. 2006) subsequently found that human airway cells are susceptible to avian as well as human influenza viruses, albeit that different cell types are infected. The latter finding emphasizes that the hypothesis stating that demonstration of both receptors in a single host will support reassortment, is no longer accurate. Indeed, presence of both receptors on a single host cell is a requirement for such reassortment events to occur.
Key FLUPIG data in view of the receptor hypothesis:
- The pig-passaged avian H1N1 virus had acquired several mutations in the receptor-binding site of the HA that switch receptor-binding specificity to a preference for the human-type (2-6 Sia) instead of the avian-type receptor (2-3 Sia). However, this in itself was insufficient to make the virus “swine-adapted”.
- The wholly avian H9N2 virus has a leucine at position 226 of the HA, which is known to make the virus bind better to the human-type receptor than related H9N2 viruses with a glutamine at this position. Despite this supposed “advantage” we could also not obtain a fully swine-adapted virus.
- Studies of the pH optimum for fusion of influenza viruses in culture suggest that this characteristic is another important determinant of viral host range restrictions. The HA of “swine-adapted” viruses has a higher pH optimum of fusion and is less stable than that of avian viruses or known pandemic viruses.
The role of the pig as a mixing vessel for genetic reassortment
Many viruses circulating in swine in Europe and the US are reassortants or even multiple reassortants with genetic contents from three different progenitor viruses. For a period of time, it has been believed that pigs are the sole mixing vessel in which reassortment events can occur. However, the data regarding the role of pigs as mixing vessel are conflicting. For example, in the 1957 and 1968 pandemics in humans, which were both caused by reassortants between the circulating human influenza virus of that time and an avian virus, there is in fact no direct evidence for the role of pigs in the generation of the reassortant viruses. On the other hand, the 2009 pandemic H1N1 influenza virus from Mexico is clearly derived from viruses that have been circulating in pigs for at least ten years and there is no doubt that a reassortment event has led to the novel H1N1 pandemic. Six of the eight gene segments were derived from a triple reassortant virus that had been circulating in swine in North America and 2 segments were derived from a Eurasian SIV (Garten et al. 2009). However, before its detection in humans in the spring of 2009, viruses with identical gene constellation have never been reported in swine anywhere in the world, although this may have been caused by a lack of proper surveillance.
Another question that remains unanswered so far is which reassortant events are required to obtain subsequent human-to-human transmission. It has long been known that SIVs sporadically jump to humans. Indeed, there are some 70 documented cases of swine flu in humans since 1958 and almost any of the established North American or European SIV genotypes has occasionally been isolated from humans. Most of these people had close contact with pigs. Importantly, all of the SIVs so far lacked the critical property to spread further in the human population until the recent outbreak with the novel 2009 H1N1 pandemic virus. The latter did acquire the unique capacity to spread among humans.
In conclusion, the novel H1N1 pandemic virus points out that we still do not fully understand the dynamics of reassortment events of influenza viruses.
Key FLUPIG data in view of reassortment:
- Reassortant influenza viruses that were created in the lab (i.e. viruses in which specific gene segments had been exchanged) were repeatedly shown to have an altered behavior as compared to the parental viruses in in vivo pig infection and transmission experiments as well as in vitro.
- On the other hand the process of genetic reassortment was difficult to reproduce in in vivo co-infections experiments in the pig. Co-infection of pigs with a swine H3N2 virus and an avian H1N2 virus predominantly resulted in viruses with an H3N2 genotype, confirming the replication advantage of the swine influenza virus over the avian virus. Similarly, we did not succeed in creating a 2009 pH1N1-like virus by concurrent inoculation of pigs with its supposed predecessors, a North America triple reassortant H1N1 virus and a European avian-like H1N1 virus.
Transmission of influenza viruses between animal species
From the previous chapters it is clear that transmission of influenza virus can occur from one species to another. However, transmission to a new host per se is not sufficient to result in a pandemic. For example, it was found that none of the “fully” avian viruses that were transmitted from bird to pig, as described previously, were able to maintain themselves in the pig population. Thus, once introduced into the pig population, the fully avian virus lacks the capacity to establish a full replication potential in pigs (De Vleeschauwer et al. 2009, 2010) and to be further transmitted between pigs. Similarly, most SIVs that are transmitted from pig to human do not become established in the human population. A subsequent adaptation is required. That such adaptation can occur is without debate, as various avian-like lineage viruses subsequently became established in the pig population (Pensaert et al. 1981; Scholtissek et al. 1983; Donatelli et al. 1991; Schultz et al. 1991; Brown et al. 1997; Webby and Webster 2001).
As for the susceptibility, a range of viral as well as host factors will most likely determine whether an influenza virus is transmitted and whether it is subsequently maintained in the population.
Key FLUPIG data in view of transmission:
- The adaptation of avian influenza viruses to pigs seems to progressively reduce the infectivity of the virus for poultry species that are highly susceptible for avian influenza such a s turkeys. Similarly, early isolates of the European avian-like H1N1 swine influenza virus efficiency replicated in and transmitted between turkeys. In contrast, more recent isolates of the avian-like H1N1 swine influenza virus lineage either failed to replicate in turkeys or showed a reduced fitness.
- See also in first paragraph on adaptation.
Immune response upon SIV infection in pigs
An infection with SIV induces a rapid and efficient immune response, which results in complete elimination of the virus within a week and a solid protection against reinfection. The specific immune response to SIV includes the production of antibodies in the circulation and at the mucosae of the respiratory tract, as well as a cell-mediated immune (CMI) response (Larsen et al. 2000; Heinen et al. 2000; Heinen et al. 2001). Antibodies are produced to all the major viral proteins (reviewed by Cox et al. (Cox et al. 2004)). Antibodies to the surface glycoproteins, HA and NA, are associated with resistance to infection, whereas antibodies to the conserved internal antigens, M and NP, are not protective. While anti-NA antibodies hamper the release of newly formed virus from infected cells, only antibodies to the globular head region of the HA can neutralize the virus and thus prevent an infection. These antibodies can be detected in virus neutralization (VN) or haemagglutination inhibition (HI) assays. The HA-specific antibodies are most efficient in terms of protection, but they are also subtype- and to some extent strain-specific. Cytotoxic T lymphocytes (CTLs) are mainly directed to the internal NP and M proteins, which are highly conserved between influenza viruses. CTLs mainly play a role in virus clearance and recovery from illness. The cytotoxic T lymphocyte (CTL) response may confer a more broad-spectrum protection, but it must be said that its protective capacity has never been demonstrated in swine.
There are only limited data about the extent of cross-protection between influenza virus variants of the same subtype or between different influenza virus subtypes in man or in swine. Because of the complex evolution and the regional differences, SIVs belonging to the same HA subtype frequently show substantial genetic and antigenic differences in their HA. The degree of homology in the amino acid sequence of the H1 of the dominant European swine H1N1 and H1N2 viruses, for example, is only 70% (Van Reeth et al. 2004). Similarly, both viruses show less than 75% homology with the viruses with a classical H1, which are widespread in North America. These differences are as large as those seen between some HA subtypes, such as H2 and H5, and they explain the lack of serological cross-reactivity between these viruses in cross-HI assays. All is illustrated by, for example, the HA of the recently emerged novel 2009 H1N1 pandemic virus. The HA of this virus is relatively closely related to the North American swine viruses with a classical H1, but very different from European H1 SIVs or the seasonal human H1N1 viruses. Some researchers therefore consider the introduction of the pandemic virus in the human population a “pseudo”-antigenic shift (Gatherer 2009).
It is generally accepted that there is no cross-protection between influenza viruses of an entirely different HA subtype. Some degree of cross-protection, however, has been demonstrated in experiments with European H1N1, H3N2 and H1N2 SIVs. When pigs with infection immunity to one of the 3 subtypes were challenged with a second subtype 4 weeks later, virus excretion was on average one or two days shorter than in influenza-naïve control pigs and disease was milder (Van Reeth et al. 2003a). Pigs with infection-immunity to the European H1N1 SIV were also largely protected against challenge with a low pathogenic H5N1 avian influenza virus (AIV) (Van Reeth et al. 2009). This type of protection was associated with cross-reactive antibodies to the N1 neuraminidase, as well as with cross-reactive CMI responses. In very recent experiments, the SIV group of UGent has demonstrated cross-protection between H1N1 SIVs circulating in Europe (avian-like H1N1) and North America (triple reassortant H1N1), despite the absence of detectable cross-reactive HI antibodies (Van Reeth et al., unpublished data).
In contrast to an infection with live influenza virus, the inactivated vaccines that are used in swine and humans induce a less solid and versatile immune response ((Van Reeth et al. 2003b). The true efficacy of such vaccines against antigenic variants of influenza, and the underlying immune mechanisms have been poorly studied.
Key FLUPIG data in view of immunity:
- Both heterovariant and heterosubtypic immunity between swine influenza viruses were studied by challenge studies in pigs. These studies showed that past-infection immunity may offer substantial cross-lineage protection against viruses of the same HA and/or NA subtype. That is, there was substantial though differing cross-protection between the 2009 pH1N1 virus and other H1 viruses. This heterovariant protection was directly correlated with the relatedness in the viral HA and NA proteins. On the other hand, true heterosubtypic protection (i.e. protection between H1 and H3 viruses) was very weak. Cross-protection between human seasonal H3N2 virus and the 2009 pH1N1 virus was studied in the mouse model. Mice that had been previously infected with the H3N2 virus displayed reduced weight loss after challenge infection and cleared the 2009 pH1N1 virus more rapidly. Adoptive transfer experiments revealed that virus-specific CD8+ T cells in concert with CD4+ T cells were responsible for the observed protection.
- The consortium also developed two novel vaccine technologies for swine influenza virus: 1) a vector vaccine in which a portion of the genome of the porcine herpesvirus pseudorabies was deleted to attenuate the virus and to create space to insert the genes encoding the influenza virus surface proteins HA o NA, 2) live vaccines that were attenuated by reducing the length of the NS1 protein of influenza virus, or by modifying the cleavage site in the influenza viral HA to require elastase enzyme. Both vaccines gave promising results in challenge studies with the homologous virus included in the vaccine and their ability to protect against more diverse viruses should be examined in the future.
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28. Van Reeth, K. et al. (2004). "Genetic relationships, serological cross-reaction and cross-protection between H1N2 and other influenza A virus subtypes endemic in European pigs." Virus Res 103(1-2): 115-124.
29. Gatherer, D. (2009). "The 2009 H1N1 influenza outbreak in its historical context." J Clin Virol 45(3): 174-178.
30. Van Reeth, K. et al. (2003a). "Protection against a European H1N2 swine influenza virus in pigs previously infected with H1N1 and/or H3N2 subtypes." Vaccine 21(13-14): 1375-1381.
31. Van Reeth, K. et al. (2009). "Prior infection with an H1N1 swine influenza virus partially protects pigs against a low pathogenic H5N1 avian influenza virus." Vaccine 27(45): 6330-6339.
32. Van Reeth, K. et al. (2003b). "Investigations of the efficacy of European H1N1- and H3N2-based swine influenza vaccines against the novel H1N2 subtype." Vet Rec 153(1): 9-13.
Potential Impact:
Impact, dissemination and exploitation
As stated in Articles 3 and 46 of the “Rules for Participation” (RfP) and Articles II.9 and II.30 of the “EC model Grant Agreement” (ECGA), a key objective of the participants in FLUPIG was to ensure swiftly and maximal dissemination of the knowledge and other foreground generated in the project. Therefore, all partners agreed on a strategy for the use and dissemination of knowledge generated by FLUPIG, as defined in the Consortium Agreement.
Dissemination
Internal dissemination
Internal meetings and reports
Internal dissemination of knowledge generated by FLUPIG was ensured by work package team meetings and formal internal progress meetings, as well as by additional meetings when requested by one or more of the participants. In total, 5 FLUPIG progress meetings and 3 teleconferences with the entire consortium were held throughout the project period. Each meeting or teleconference was documented in minutes that, subsequently, were shared between the participants. Smaller scale teleconferences with a restricted number of partners were held regularly to discuss specific WPs or tasks. Three formal periodic reports on the scientific and technological results of the project were delivered to the relevant EC Commission body.
Website
A website was designed within the first three months after kick-off. The website has been regularly updated with results and relevant information. Several parts of the website are confidential (password-protected) and accessible to FLUPIG members only to ensure protection of the information provided. This information includes but is not limited to meeting reports, presentations, agreements, import permits and other shipment details as well as standard operating procedures.
Training
During the project, students and/or co-workers of four partner institutes followed training stays in participant laboratories in order to obtain an expertise at different levels (e.g. molecular and cell biological virology, immunology, animal experimentation, ex vivo organ cultures, ...). A summary of these training visits is provided in the Project periodic reports.
External dissemination
Taking the entire FLUPIG reporting period into account, the Consortium has accomplished 105 dissemination activities. A more detailed description of the dissemination activities is provided below and on the Participant Portal (“Dissemination Activities”).
Website
As mentioned above, a website was designed and parts of these website are public. This way, influenza researchers, veterinarians and health care workers outside the consortium are informed rapidly about major findings of FLUPIG. Information provided on the public website includes a project summary, contact details of FLUPIG partner institutes, references to publications and photo’s of annual meetings.
Scientific publications
All participants took care of the scientific output of this project via the usual routes, such as publications, presentations, participation in discussion groups. In order to facilitate the identification of the knowledge by the public and the Commission, disseminated knowledge and other foreground concerning results from FLUPIG were marked with the project name and with the grant agreement number 258084 (Article 45 RfP – Article II.30.4 of ECGA).
During the entire FLUPIG project, a total of 35 scientific papers were published in international peer-reviewed journals and 1 scientific publication in the journal of the Pig Veterinary Society.
Other scientific meetings
As many scientists were overwhelmed by travel obligations, the FLUPIG consortium decided not to organize international scientific meetings by itself. Rather, it was decided to actively participate in international conferences related to influenza and one health to ensure dissemination of results to a broad audience of scientists without additional travel requirements. In The FLUPIG project and/or its detailed results were presented at many national and international scientific conferences either as oral poster presentations. The coordinator also participated in the EFSA “Workshop on Research Gap Analysis in Animal Influenza” held in Parma on January 8th and 9th 2015. In addition, several FLUPIG partners will participate in and present their most recent FLUPIG data at the third “Neglected Influenza Virus Symposium” in Athens, Georgia, April 16th-17th 2015. This meeting focuses on influenza in species others than human and birds, swine influenza in particular.
Interactions with institutions and/or universities involved in influenza research and control
The FLUPIG consortium interacted with:
− Other scientific project teams addressing influenza specific topics. These included ESNIP3 (FP7), FLURISK (EFSA), NADIR (FP7). The fact that several partners of the FLUPIG consortium also participated in the above mentioned projects facilitated the interaction.
− Scientists working in the public health and medical fields as well as with public health authorities. Data on the overall ecology of SIVs and their potential relevance for other animal species, in particular human beings, were shared with swine influenza reseach worldwide and members of the OFFLU SIV group in particular.
− National Reference Laboratories, the Food and Agriculture Organization of the United Nations (FAO), the European Food Safety Authority (EFSA) and the World Organization for Animal Health (OIE). The fact that several representatives of these organizations were part of the FLUPIG consortium (P1, 2, 4, 5, 6, 7 and 10) facilitated this networking part.
Education
Several participants (P1, P3, P5, P6, P8, and P9) of FLUPIG are involved in teaching of students at universities. This involves lectures in basic virology, viral diseases of swine and humans and viral zoonoses. Furthermore, many of the participants are involved in local veterinary training initiatives.
Dissemination to end-users
End-users included, but were not limited to veterinarians, human health care workers, swine farmers, and, importantly, the society. Recently, the zoonotic potential of SIVs has clearly been highlighted with the worldwide outbreak of pandemic (H1N1) 2009 influenza virus as well as with the outbreak of the H3N2 variant virus in the USA. Our work could contribute to a more rational public health risk analysis of swine influenza by communicating our most important findings to human influenza networks, to the WHO, EFSA, the Centers for Disease Control and Prevention (CDC), the ECDC, and to the media.
Dissemination to end-users was achieved by a scientific paper published in the journal of the Pig Veterinary Society, as well as lectures for swine producers and veterinarians.
Exploitation
Various approaches were used to design and test experimental live-attenuated influenza virus and a multivalent PrV/influenza vaccines in swine. Both vaccine approaches proved effective in experimental settings and will be further explored for their potential commercial application.
No other (potential) exploitable results were generated throughout the project.
Management of intellectual property
As recommended by the Commission, Intellectual Property Rights (IPR) issues were dealt with in an early stage of the project. Basic legal requirements as well as a FLUPIG-dedicated MTA were included in the Consortium Agreement. Arrangements reflected EC policy and guidelines related to IPR and exploitation of results within a EC-funded project. This included the RfP, ECGA, and “DESCA – The simplified FP7 model Consortium Agreement. www.desca-FP7.eu”. The Consortium Agreement was negotiated with and signed by all participants with a slight delay of 5 months. The delay had no impact on other management tasks, on scientific tasks or on the use of resources.
No decisions were to be made on major knowledge management issues such as patenting, licensing and other exploitations of project results.
List of Websites:
http://www.flupig.ugent.be
The FLUPIG project aimed to gain better insights into the role of pigs in the generation of novel pandemic influenza viruses for humans. We have performed in vivo infection studies with avian H1N1 and H9N2 influenza viruses in pigs, ferrets and birds with the purpose to identify factors that mediate adaptation of avian influenza viruses to pigs (WP 1), as well as transmission of these viruses between pigs (WP 2) and from pigs to other animal species (WP 3). All three steps would be required to turn a wholly avian virus into a pandemic virus. The process of adaptation of avian influenza viruses to pigs (WP 1) was studied both at the level of the host and virus. The swine host was found to be similar to humans in some aspects, but not in other. For example, the prevalence and distribution of “avian-type” and “human-type” influenza virus receptors in the airways appeared to be almost identical in pigs and in humans. The avian-type receptor was very scarce in the nasal mucosa and trachea and largely restricted to the lungs, whereas the human-type receptor was found throughout the respiratory tract. However, there appeared to be differences between pigs and humans in the stage of virus replication that follows attachment of the virus to host receptors. Indeed, the pH optimum at which the viral surface protein hemagglutinin (HA) undergoes a conformational change that triggers the so-called fusion of the virus with the host cell was found to be higher for swine than for human influenza viruses. We used two different approaches to try to adapt avian influenza viruses to pigs: a) blind serial passages with wholly avian influenza viruses in the pig, with the purpose to introduce mutations that make the virus more fit to replicate in swine and b) genetic “re-assortment” in the lab, resulting in a hybrid virus with surface protein genes of the avian H9N2 virus and internal protein genes of the 2009 pandemic H1N1 virus. The latter virus is known to be well adapted for replication in both swine and humans. Both approaches resulted in a slight to moderate enhancement of avian influenza virus replication in the pig, but they were insufficient to make the avian viruses fully swine adapted. Though the pig-passaged or reassortant viruses were able to transmit between pigs, transmission remained highly inefficient when compared to that of natural swine adapted viruses. Genetic analyses of pig-passaged viruses revealed several mutations in the HA as well as other viral genes. Remarkably, replication efficiency and transmission of the wholly avian H1N1 influenza virus was much better in ferrets than in pigs, which suggests that the pig and ferret present different species barriers. The question as to whether viruses with an H9, which have not become established in the swine or human population so far, may ever become adapted to pigs as H1 and H3 did remained unanswered. While it is possible that a different or more forced approach would result in fully swine adapted viruses, adaptation of avian viruses to the pig clearly does not occur easily. Interestingly, adaptation of a duck H1N1 virus to pigs made the virus less fit for replication in birds. Pandemic influenza viruses must not only have the ability to spread efficiently from person to person, another requirement is that immunity within the human population must be sufficiently low to allow widespread infection. Studies in pigs and/or mice showed substantial cross-protection between antigenically distinct influenza viruses within a given HA subtype, but only limited cross-protection between viruses with a different HA subtype (WP 4). The consortium also developed novel live vaccines for swine influenza virus that were stably attenuated by biotechnology. Both vaccines gave promising results in challenge studies with the homologous virus included in the vaccine and their ability to protect against more diverse viruses should be examined in the future.
Project Context and Objectives:
Influenza A viruses originate in wild birds and are transmitted to other avian and mammalian species (including humans) in which they may cause self-limiting infections, epidemics, or panzootics. A few times each century they cause pandemics. The mechanisms by which influenza viruses gain the capacity of abandoning the animal reservoir and becoming widespread in human beings are currently unknown.
Among all animals, the pig is believed to play an essential role in the influenza virus ecology, since it has been shown that (1) pigs are susceptible to all subtypes of influenza A viruses, including those of avian origin; (2) pigs have receptors for both avian and mammalian origin viruses, and thus represent an ideal vessel for viral reassortment or adaptation of an avian virus to the mammalian host. In fact, influenza viruses isolated from swine often contain genes, which belong to viruses isolated from other species. The most recent example is the 2009 H1N1 pandemic virus, which has originated from swine herds in North America. It is a reassortant, which contains genes which can be traced back to three species (avian, human and swine) and two hemispheres. Six gene segments are similar to ones previously found in triple reassortant swine influenza viruses (SIVs) circulating in pigs in North America, from which the pandemic virus has derived a classical H1 haemagglutinin (HA). Of these 6 genes, three (HA, nucleoprotein (NP), non-structural protein (NS)) had been established in viruses endemic in the pig population for decades, Polymerases PB2 and PA were introduced into pigs from the avian host in 1998 and the PB1 appears to be derived from a human virus introduced in 1998. The two other gene segments, encoding the N1 neuraminidase (NA) and matrix proteins (MP) are derived from a virus circulating in swine populations in Europe or Asia. Thus, this virus has a core of genes that had been circulating in SIVs for over 30 years, which has acquired in more recent times two avian genes and one human gene.
It should be stressed however, that there is no direct evidence that pigs have played a role in the genesis of any of the three pandemics of the 20th century. In other words, the hypothesis of the pig as an intermediate host between birds and humans has never been proven for any of the pandemic viruses of the last century. In fact, the swine-human link for both the 1957 and 1968 pandemic viruses (both of which were reassortants between the human virus circulating at the time and an avian virus of a novel HA subtype) has not been established.
Both in the current and last century there have also been documented episodes of transmission of influenza virus from swine to humans, although these SIVs failed to spread further in the human population. The reason for this is unclear. In addition, as far as we know, most SIVs of the H1N1, H3N2 and H1N2 subtypes are endemic in the swine population and only sporadically cause human infection.
Though the pandemic (H1N1) 2009 influenza virus almost certainly comes from pigs, it is still unclear why and how this novel H1N1 virus obtained the capacity for human-to-human spread, while the established SIVs have so far failed to do so. On the other hand, we have evidence that all known pandemic viruses contain an avian genetic component – and it is unclear what role is played by genes of avian origin in the generation of pandemic viruses, particularly in view of the widespread infection of poultry with H5N1 and H9N2 viruses. Co-circulation of the pandemic (H1N1) 2009 influenza virus and other animal influenza viruses (avian H5N1 and H9N2 and swine H3N2, H1N1 and H1N2) in susceptible hosts could represent the occasion for viral reassortment with unpredictable consequences.
Another aspect that is to be considered is that pigs support the endemic circulation of viruses that belong to the same subtype of viruses that have been shown (to date) to cause pandemics in humans. Understanding how the immune system of pigs responds to infection and the extent of cross-protection within and between virus subtypes is crucial to limiting the degree of circulation of these viruses in pigs, as is the development of novel vaccination strategies.
Though obviously a rare event, there is a real danger that swine-origin reassortants start to spread readily between humans and launch a pandemic, and this is perfectly illustrated by the novel H1N1 virus. In order to proactively respond to such threats in the future, we will need to fill in the lack of knowledge about the role of pigs in overall influenza ecology. In order to do so, following questions have to be addressed:
Q1. What are the mechanisms whereby an avian virus is able to adapt in the pig host and subsequently establish a new lineage in pigs, and what is the true significance of pigs as a mixing vessel?
Q2. What determines the transmissibility of influenza virus between pigs?
Q3. What determines the transmissibility of influenza virus from pigs to other relevant animal species, including ferrets as a model for humans?
Q4. What determines the transmissibility of influenza virus from ferret to ferret as a model for human-to-human transmission?
Q5. Can previous infections or vaccinations with influenza virus protect against novel, antigenically different viruses?
The FLUPIG consortium aimed to study each of these questions based on its solid experience in influenza pathogenesis and transmission in a wide variety of ex vivo models as well as in vivo animal models, including swine. In particular, we aimed at improving our knowledge on the gene constellation and genetic interactions that are necessary to generate pandemic viruses in combination with an improved understanding of the host-dependent variables such as receptor distribution and immune response. Gaining insight in the characteristics of the pathogen and combining this with the host component will be the main goal of our consortium. Combined with improved surveillance for influenza in animals, effective vaccines and antiviral drugs, this knowledge will be critical to the control of future influenza pandemics.
The specific objectives of the consortium were:
Study the replication efficiency of different viruses in pigs in vivo, as well as in reliable in vitro models
− Identify specific viral amino acid signatures and gene constellations that enhance the replication of influenza viruses in pigs;
− Determine whether viruses with an H9, which have not become established in the swine or human population so far, may ever become adapted in pigs as H1 and H3 did;
− Gain insights into the host factors, including virus receptors, that contribute to the replication efficiency of influenza viruses in pigs.
Study the transmissibility of different viruses between pigs
− Investigate mechanisms whereby non-swine HA and NA restrict virus transmissibility in pigs;
− Determine the genetic factors that influence virus transmissibility and pathogenesis of the pandemic (H1N1) 2009 influenza virusin pigs;
− Identify genetic changes in influenza viruses necessary for acquisition of swine and human transmissibility;
− Compare virus transmissibility in the swine model with that of ferrets and in vitro/ex vivo tools.
Study the transmissibility of different viruses from pigs to other relevant animal species
− Investigate and compare the capabilities of SIVs to be transmitted from pigs to other relevant animal species, with particular reference to birds;
− Investigate and compare the capabilities of SIVs to be transmitted from pigs to ferrets, as a representative model for humans;
− Determine the role of selected swine influenza virus (SIV) genetic determinants for inter-species transmission.
To study heterovariant and heterosubtypic cross-protection between influenza viruses in pigs
− To identify the level of cross-protective immunity between H1N1 viruses with an antigenically different H1 (heterovariant protection) (e.g. European swine H1N1 virus versus novel 2009 H1N1 pandemic virus; human seasonal H1N1 viruses versus novel 2009 H1N1 pandemic virus);
− To identify the level of cross-protective immunity between SIVs belonging to different HA subtypes or between SIVs and avian influenza viruses (AIVs) (heterosubtypic protection);
− To identify the immune mechanisms mediating cross-protection between influenza viruses;
− To evaluate novel modified-live attenuated vaccine (MLV) candidates against swine influenza.
Project Results:
Scientific and technical results and foreground
For a long period of time, there is the hypothesis that pigs play a key role in the development of influenza viruses with pandemic potential. Several arguments were raised to support that hypothesis, but review of historical data indicated that the hypothesis is an oversimplification. Therefore, the FLUPIG consortium aimed at gaining more insight in the role of the pig by addressing several of the traditional assumptions in experimental studies. An overview of the results obtained and their relevance in view of the role of the pig are briefly outlined below.
The susceptibility of pigs to influenza viruses of avian origin: the species barrier
Historical data point out that direct avian-to-pig transmission of influenza viruses regularly occurred under natural conditions (Pensaert et al. 1981; Peiris et al. 2001; Guan et al. 1996; Loeffen et al. 2004). Furthermore, experiments performed by Kida and colleagues (Kida et al. 1994) proved that pigs are receptive to a wide variety of AIVs, whereas an earlier experimental study in human volunteers showed that humans were largely refractory to infection with certain AIVs (Beare and Webster 1991). However, while the pigs in the study of Kida et al. were fully influenza-naïve, most human volunteers had previously been infected with influenza viruses, so it cannot be excluded that this affected their susceptibility to AIVs.
Another argument that the unique susceptibility of pigs to AIVs is not as obvious as previously believed is provided by the recent outbreaks with a highly pathogenic avian H5N1, with an avian influenza H7N7 virus and with an avian H7N9 virus. The H5N1 outbreak is the largest and most devastating outbreak of highly pathogenic avian influenza in history, not only affecting millions of chickens and ducks, but also having a serious impact on human health with an exceptionally high case-fatality rate. However, most, if not all, of the human cases were in people with close contact with infected poultry and direct exposure to large amounts of H5N1 virus. Pigs can and do become infected with avian H5 subtypes under natural and experimental conditions, but all evidence indicates that the currently circulating H5N1 viruses are not well adapted for replication in pigs (Lipatov et al. 2008; De Vleeschauwer et al. 2009; De Vleeschauwer and Van Reeth 2010). The few available experimental infection studies indicate that the H5N1 virus replicates in the respiratory tract of pigs, but to a much lower extent than typical SIVs and mainly in the lungs. Remarkably, the pigs showed only mild or no clinical signs, while the viruses were extremely virulent for poultry. Also, the infected pigs failed to transmit the virus to uninfected littermates.
Key FLUPIG data in view of the species barrier hypothesis:
- Wholly avian H1N1 (duck/Bavaria/77) and H9N2 (quail/Hong kong/G1/97) viruses replicated in pigs, but replication was highly inefficient when compared to that of typical swine-adapted influenza viruses. Though there was limited pig-to-pig transmission of these viruses under experimental conditions, transmission as well was highly inefficient when compared to that of typical SIVs.
- Serial (up to 25), blind passages with these avian H1N1 and H9N2 viruses resulted in a slightly enhanced replication and/or transmission efficiency, but we failed to obtain fully swine adapted viruses by this adaptation. In addition, the “partially swine-adapted” H9N2 virus was lost again upon further passages.
- The pig passaged avian H1N1 viruses showed several mutations that are similar to those observed in early isolates of the “avian-like” H1N1 SIV, including mutations in the receptor-binding site that are known to enhance binding to the “human-type” receptors. However, none of the viruses acquired a full set of mutations found in natural swine viruses.
- The unadapted wholly avian H1N1 virus showed a better replication in and transmission between ferrets than in pigs. This suggests that the pig and the ferret present different barriers for HA and NA functions.
- Similarly a reverse-genetics engineered H9N2 reassortant virus, with the avian HA and NA in a backbone of a 2009 pandemic H1N1 virus, showed a slightly better replication in and transmission between pigs than the wholly avian H9N2 virus. But this approach, as well pig-passage with the reassortant virus, were also insufficient to make the avian virus fully adapted to pigs.
- In in vitro viral attachment assays, an avian H7N9 virus showed only limited attachment to the nasal mucosa of pigs. Stronger attachment was observed with nasal mucosa of human, ferrets, macaques and guinea pigs.
- Our data underscore the difficulty of reproducing virus adaptation in an experimental setting. It is possible that adaptation would have occurred with a more forced approach or with a combination of lab-made genetic changes to the viruses (e.g. reassortment in addition to specific viral mutations) and passages.
The pattern of receptor expression needed to enter into the respiratory tract
Another reason why pigs were appointed as prime candidates for the role as intermediate hosts is related to the nature of the influenza receptors in the respiratory tract. Binding of a virus to its receptor is the very first step in the viral replication cycle. For an influenza infection, binding is accomplished between the viral HA protein and the cellular sialic acid site. Although all influenza viruses recognize sialic acid, the type of sialic acid linkage with its underlying sugar residue as well as the receptor specificity of the viral HA differ between host species. AIVs preferentially bind to the sialic acid α 2,3 Gal linkage type (2-3 Sia), whereas human influenza viruses favour binding to the sialic acid α 2,6 Gal linkage type (2-6 Sia). Subsequently, it was found that the preferential replication site for influenza in ducks expressed α 2,3 Gal (Ito et al. 1998), whereas the human respiratory tract mainly expressed the α 2,6 Gal (Baum and Paulson 1990). As the pig trachea was found to contain both receptors, it was easily concluded that the pig could allow (1) adaptation of the virus from avian-type to human-type receptors providing a potential link from birds to humans (Ito et al. 1998; Ito 2000) and/or (2) simultaneous infection of a host cell with an avian and human virus and subsequent genetic reassortment.
More recent data showed that the sialic acid expression in the various host species is not as straightforward as initially thought. In fact, Van Poucke et al. (Van Poucke et al. 2010) could not confirm the studies of Ito et al., (1998). They found moderate to abundant expression of the human-like virus receptors in nasal, tracheal, bronchial and lung explants of pigs, but only a minimal expression of the avian-like receptors, especially in the nasal, tracheal and bronchial epithelium. The lack of α 2,3 Gal receptor expression correlated well with a lack of avian virus replication. The ex vivo studies of Van Poucke and colleagues are in agreement with the virus-binding experiments of van Riel et al. (van Riel et al. 2007). Additional studies of the human respiratory tract demonstrated a receptor distribution pattern similar to that in pigs with the presence of both sialic acid types (Shinya et al. 2006). Matrosovich et al. (Matrosovich et al. 2004) and Thompson et al. (Thompson et al. 2006) subsequently found that human airway cells are susceptible to avian as well as human influenza viruses, albeit that different cell types are infected. The latter finding emphasizes that the hypothesis stating that demonstration of both receptors in a single host will support reassortment, is no longer accurate. Indeed, presence of both receptors on a single host cell is a requirement for such reassortment events to occur.
Key FLUPIG data in view of the receptor hypothesis:
- The pig-passaged avian H1N1 virus had acquired several mutations in the receptor-binding site of the HA that switch receptor-binding specificity to a preference for the human-type (2-6 Sia) instead of the avian-type receptor (2-3 Sia). However, this in itself was insufficient to make the virus “swine-adapted”.
- The wholly avian H9N2 virus has a leucine at position 226 of the HA, which is known to make the virus bind better to the human-type receptor than related H9N2 viruses with a glutamine at this position. Despite this supposed “advantage” we could also not obtain a fully swine-adapted virus.
- Studies of the pH optimum for fusion of influenza viruses in culture suggest that this characteristic is another important determinant of viral host range restrictions. The HA of “swine-adapted” viruses has a higher pH optimum of fusion and is less stable than that of avian viruses or known pandemic viruses.
The role of the pig as a mixing vessel for genetic reassortment
Many viruses circulating in swine in Europe and the US are reassortants or even multiple reassortants with genetic contents from three different progenitor viruses. For a period of time, it has been believed that pigs are the sole mixing vessel in which reassortment events can occur. However, the data regarding the role of pigs as mixing vessel are conflicting. For example, in the 1957 and 1968 pandemics in humans, which were both caused by reassortants between the circulating human influenza virus of that time and an avian virus, there is in fact no direct evidence for the role of pigs in the generation of the reassortant viruses. On the other hand, the 2009 pandemic H1N1 influenza virus from Mexico is clearly derived from viruses that have been circulating in pigs for at least ten years and there is no doubt that a reassortment event has led to the novel H1N1 pandemic. Six of the eight gene segments were derived from a triple reassortant virus that had been circulating in swine in North America and 2 segments were derived from a Eurasian SIV (Garten et al. 2009). However, before its detection in humans in the spring of 2009, viruses with identical gene constellation have never been reported in swine anywhere in the world, although this may have been caused by a lack of proper surveillance.
Another question that remains unanswered so far is which reassortant events are required to obtain subsequent human-to-human transmission. It has long been known that SIVs sporadically jump to humans. Indeed, there are some 70 documented cases of swine flu in humans since 1958 and almost any of the established North American or European SIV genotypes has occasionally been isolated from humans. Most of these people had close contact with pigs. Importantly, all of the SIVs so far lacked the critical property to spread further in the human population until the recent outbreak with the novel 2009 H1N1 pandemic virus. The latter did acquire the unique capacity to spread among humans.
In conclusion, the novel H1N1 pandemic virus points out that we still do not fully understand the dynamics of reassortment events of influenza viruses.
Key FLUPIG data in view of reassortment:
- Reassortant influenza viruses that were created in the lab (i.e. viruses in which specific gene segments had been exchanged) were repeatedly shown to have an altered behavior as compared to the parental viruses in in vivo pig infection and transmission experiments as well as in vitro.
- On the other hand the process of genetic reassortment was difficult to reproduce in in vivo co-infections experiments in the pig. Co-infection of pigs with a swine H3N2 virus and an avian H1N2 virus predominantly resulted in viruses with an H3N2 genotype, confirming the replication advantage of the swine influenza virus over the avian virus. Similarly, we did not succeed in creating a 2009 pH1N1-like virus by concurrent inoculation of pigs with its supposed predecessors, a North America triple reassortant H1N1 virus and a European avian-like H1N1 virus.
Transmission of influenza viruses between animal species
From the previous chapters it is clear that transmission of influenza virus can occur from one species to another. However, transmission to a new host per se is not sufficient to result in a pandemic. For example, it was found that none of the “fully” avian viruses that were transmitted from bird to pig, as described previously, were able to maintain themselves in the pig population. Thus, once introduced into the pig population, the fully avian virus lacks the capacity to establish a full replication potential in pigs (De Vleeschauwer et al. 2009, 2010) and to be further transmitted between pigs. Similarly, most SIVs that are transmitted from pig to human do not become established in the human population. A subsequent adaptation is required. That such adaptation can occur is without debate, as various avian-like lineage viruses subsequently became established in the pig population (Pensaert et al. 1981; Scholtissek et al. 1983; Donatelli et al. 1991; Schultz et al. 1991; Brown et al. 1997; Webby and Webster 2001).
As for the susceptibility, a range of viral as well as host factors will most likely determine whether an influenza virus is transmitted and whether it is subsequently maintained in the population.
Key FLUPIG data in view of transmission:
- The adaptation of avian influenza viruses to pigs seems to progressively reduce the infectivity of the virus for poultry species that are highly susceptible for avian influenza such a s turkeys. Similarly, early isolates of the European avian-like H1N1 swine influenza virus efficiency replicated in and transmitted between turkeys. In contrast, more recent isolates of the avian-like H1N1 swine influenza virus lineage either failed to replicate in turkeys or showed a reduced fitness.
- See also in first paragraph on adaptation.
Immune response upon SIV infection in pigs
An infection with SIV induces a rapid and efficient immune response, which results in complete elimination of the virus within a week and a solid protection against reinfection. The specific immune response to SIV includes the production of antibodies in the circulation and at the mucosae of the respiratory tract, as well as a cell-mediated immune (CMI) response (Larsen et al. 2000; Heinen et al. 2000; Heinen et al. 2001). Antibodies are produced to all the major viral proteins (reviewed by Cox et al. (Cox et al. 2004)). Antibodies to the surface glycoproteins, HA and NA, are associated with resistance to infection, whereas antibodies to the conserved internal antigens, M and NP, are not protective. While anti-NA antibodies hamper the release of newly formed virus from infected cells, only antibodies to the globular head region of the HA can neutralize the virus and thus prevent an infection. These antibodies can be detected in virus neutralization (VN) or haemagglutination inhibition (HI) assays. The HA-specific antibodies are most efficient in terms of protection, but they are also subtype- and to some extent strain-specific. Cytotoxic T lymphocytes (CTLs) are mainly directed to the internal NP and M proteins, which are highly conserved between influenza viruses. CTLs mainly play a role in virus clearance and recovery from illness. The cytotoxic T lymphocyte (CTL) response may confer a more broad-spectrum protection, but it must be said that its protective capacity has never been demonstrated in swine.
There are only limited data about the extent of cross-protection between influenza virus variants of the same subtype or between different influenza virus subtypes in man or in swine. Because of the complex evolution and the regional differences, SIVs belonging to the same HA subtype frequently show substantial genetic and antigenic differences in their HA. The degree of homology in the amino acid sequence of the H1 of the dominant European swine H1N1 and H1N2 viruses, for example, is only 70% (Van Reeth et al. 2004). Similarly, both viruses show less than 75% homology with the viruses with a classical H1, which are widespread in North America. These differences are as large as those seen between some HA subtypes, such as H2 and H5, and they explain the lack of serological cross-reactivity between these viruses in cross-HI assays. All is illustrated by, for example, the HA of the recently emerged novel 2009 H1N1 pandemic virus. The HA of this virus is relatively closely related to the North American swine viruses with a classical H1, but very different from European H1 SIVs or the seasonal human H1N1 viruses. Some researchers therefore consider the introduction of the pandemic virus in the human population a “pseudo”-antigenic shift (Gatherer 2009).
It is generally accepted that there is no cross-protection between influenza viruses of an entirely different HA subtype. Some degree of cross-protection, however, has been demonstrated in experiments with European H1N1, H3N2 and H1N2 SIVs. When pigs with infection immunity to one of the 3 subtypes were challenged with a second subtype 4 weeks later, virus excretion was on average one or two days shorter than in influenza-naïve control pigs and disease was milder (Van Reeth et al. 2003a). Pigs with infection-immunity to the European H1N1 SIV were also largely protected against challenge with a low pathogenic H5N1 avian influenza virus (AIV) (Van Reeth et al. 2009). This type of protection was associated with cross-reactive antibodies to the N1 neuraminidase, as well as with cross-reactive CMI responses. In very recent experiments, the SIV group of UGent has demonstrated cross-protection between H1N1 SIVs circulating in Europe (avian-like H1N1) and North America (triple reassortant H1N1), despite the absence of detectable cross-reactive HI antibodies (Van Reeth et al., unpublished data).
In contrast to an infection with live influenza virus, the inactivated vaccines that are used in swine and humans induce a less solid and versatile immune response ((Van Reeth et al. 2003b). The true efficacy of such vaccines against antigenic variants of influenza, and the underlying immune mechanisms have been poorly studied.
Key FLUPIG data in view of immunity:
- Both heterovariant and heterosubtypic immunity between swine influenza viruses were studied by challenge studies in pigs. These studies showed that past-infection immunity may offer substantial cross-lineage protection against viruses of the same HA and/or NA subtype. That is, there was substantial though differing cross-protection between the 2009 pH1N1 virus and other H1 viruses. This heterovariant protection was directly correlated with the relatedness in the viral HA and NA proteins. On the other hand, true heterosubtypic protection (i.e. protection between H1 and H3 viruses) was very weak. Cross-protection between human seasonal H3N2 virus and the 2009 pH1N1 virus was studied in the mouse model. Mice that had been previously infected with the H3N2 virus displayed reduced weight loss after challenge infection and cleared the 2009 pH1N1 virus more rapidly. Adoptive transfer experiments revealed that virus-specific CD8+ T cells in concert with CD4+ T cells were responsible for the observed protection.
- The consortium also developed two novel vaccine technologies for swine influenza virus: 1) a vector vaccine in which a portion of the genome of the porcine herpesvirus pseudorabies was deleted to attenuate the virus and to create space to insert the genes encoding the influenza virus surface proteins HA o NA, 2) live vaccines that were attenuated by reducing the length of the NS1 protein of influenza virus, or by modifying the cleavage site in the influenza viral HA to require elastase enzyme. Both vaccines gave promising results in challenge studies with the homologous virus included in the vaccine and their ability to protect against more diverse viruses should be examined in the future.
References:
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9. De Vleeschauwer, A. and K. Van Reeth (2010). "Prior infection of pigs with swine influenza viruses is a barrier to infection with avian influenza viruses." Vet Microbiol 146(3-4): 340-345.
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Potential Impact:
Impact, dissemination and exploitation
As stated in Articles 3 and 46 of the “Rules for Participation” (RfP) and Articles II.9 and II.30 of the “EC model Grant Agreement” (ECGA), a key objective of the participants in FLUPIG was to ensure swiftly and maximal dissemination of the knowledge and other foreground generated in the project. Therefore, all partners agreed on a strategy for the use and dissemination of knowledge generated by FLUPIG, as defined in the Consortium Agreement.
Dissemination
Internal dissemination
Internal meetings and reports
Internal dissemination of knowledge generated by FLUPIG was ensured by work package team meetings and formal internal progress meetings, as well as by additional meetings when requested by one or more of the participants. In total, 5 FLUPIG progress meetings and 3 teleconferences with the entire consortium were held throughout the project period. Each meeting or teleconference was documented in minutes that, subsequently, were shared between the participants. Smaller scale teleconferences with a restricted number of partners were held regularly to discuss specific WPs or tasks. Three formal periodic reports on the scientific and technological results of the project were delivered to the relevant EC Commission body.
Website
A website was designed within the first three months after kick-off. The website has been regularly updated with results and relevant information. Several parts of the website are confidential (password-protected) and accessible to FLUPIG members only to ensure protection of the information provided. This information includes but is not limited to meeting reports, presentations, agreements, import permits and other shipment details as well as standard operating procedures.
Training
During the project, students and/or co-workers of four partner institutes followed training stays in participant laboratories in order to obtain an expertise at different levels (e.g. molecular and cell biological virology, immunology, animal experimentation, ex vivo organ cultures, ...). A summary of these training visits is provided in the Project periodic reports.
External dissemination
Taking the entire FLUPIG reporting period into account, the Consortium has accomplished 105 dissemination activities. A more detailed description of the dissemination activities is provided below and on the Participant Portal (“Dissemination Activities”).
Website
As mentioned above, a website was designed and parts of these website are public. This way, influenza researchers, veterinarians and health care workers outside the consortium are informed rapidly about major findings of FLUPIG. Information provided on the public website includes a project summary, contact details of FLUPIG partner institutes, references to publications and photo’s of annual meetings.
Scientific publications
All participants took care of the scientific output of this project via the usual routes, such as publications, presentations, participation in discussion groups. In order to facilitate the identification of the knowledge by the public and the Commission, disseminated knowledge and other foreground concerning results from FLUPIG were marked with the project name and with the grant agreement number 258084 (Article 45 RfP – Article II.30.4 of ECGA).
During the entire FLUPIG project, a total of 35 scientific papers were published in international peer-reviewed journals and 1 scientific publication in the journal of the Pig Veterinary Society.
Other scientific meetings
As many scientists were overwhelmed by travel obligations, the FLUPIG consortium decided not to organize international scientific meetings by itself. Rather, it was decided to actively participate in international conferences related to influenza and one health to ensure dissemination of results to a broad audience of scientists without additional travel requirements. In The FLUPIG project and/or its detailed results were presented at many national and international scientific conferences either as oral poster presentations. The coordinator also participated in the EFSA “Workshop on Research Gap Analysis in Animal Influenza” held in Parma on January 8th and 9th 2015. In addition, several FLUPIG partners will participate in and present their most recent FLUPIG data at the third “Neglected Influenza Virus Symposium” in Athens, Georgia, April 16th-17th 2015. This meeting focuses on influenza in species others than human and birds, swine influenza in particular.
Interactions with institutions and/or universities involved in influenza research and control
The FLUPIG consortium interacted with:
− Other scientific project teams addressing influenza specific topics. These included ESNIP3 (FP7), FLURISK (EFSA), NADIR (FP7). The fact that several partners of the FLUPIG consortium also participated in the above mentioned projects facilitated the interaction.
− Scientists working in the public health and medical fields as well as with public health authorities. Data on the overall ecology of SIVs and their potential relevance for other animal species, in particular human beings, were shared with swine influenza reseach worldwide and members of the OFFLU SIV group in particular.
− National Reference Laboratories, the Food and Agriculture Organization of the United Nations (FAO), the European Food Safety Authority (EFSA) and the World Organization for Animal Health (OIE). The fact that several representatives of these organizations were part of the FLUPIG consortium (P1, 2, 4, 5, 6, 7 and 10) facilitated this networking part.
Education
Several participants (P1, P3, P5, P6, P8, and P9) of FLUPIG are involved in teaching of students at universities. This involves lectures in basic virology, viral diseases of swine and humans and viral zoonoses. Furthermore, many of the participants are involved in local veterinary training initiatives.
Dissemination to end-users
End-users included, but were not limited to veterinarians, human health care workers, swine farmers, and, importantly, the society. Recently, the zoonotic potential of SIVs has clearly been highlighted with the worldwide outbreak of pandemic (H1N1) 2009 influenza virus as well as with the outbreak of the H3N2 variant virus in the USA. Our work could contribute to a more rational public health risk analysis of swine influenza by communicating our most important findings to human influenza networks, to the WHO, EFSA, the Centers for Disease Control and Prevention (CDC), the ECDC, and to the media.
Dissemination to end-users was achieved by a scientific paper published in the journal of the Pig Veterinary Society, as well as lectures for swine producers and veterinarians.
Exploitation
Various approaches were used to design and test experimental live-attenuated influenza virus and a multivalent PrV/influenza vaccines in swine. Both vaccine approaches proved effective in experimental settings and will be further explored for their potential commercial application.
No other (potential) exploitable results were generated throughout the project.
Management of intellectual property
As recommended by the Commission, Intellectual Property Rights (IPR) issues were dealt with in an early stage of the project. Basic legal requirements as well as a FLUPIG-dedicated MTA were included in the Consortium Agreement. Arrangements reflected EC policy and guidelines related to IPR and exploitation of results within a EC-funded project. This included the RfP, ECGA, and “DESCA – The simplified FP7 model Consortium Agreement. www.desca-FP7.eu”. The Consortium Agreement was negotiated with and signed by all participants with a slight delay of 5 months. The delay had no impact on other management tasks, on scientific tasks or on the use of resources.
No decisions were to be made on major knowledge management issues such as patenting, licensing and other exploitations of project results.
List of Websites:
http://www.flupig.ugent.be