ESNIP 2

The European Surveillance Network for Influenza in = Pigs

Swine influenza: variations = on an old=20 theme

Prof. Kristien Van Reeth
Laboratory of = Virology,=20 Faculty of Veterinary Medicine,
Ghent University, Belgium

Swine influenza (SI) is an acute viral = infection=20 of the respiratory tract and an important cause of respiratory disease = in pigs.=20 Influenza A viruses of H1N1 and H3N2 subtypes have been circulating in = the=20 European swine population for about 20 years. A swine influenza virus of = H1N2=20 subtype, however, has emerged only recently and this has complicated the = control=20 and diagnosis of SI.

One virus, multiple = subtypes=20
Swine influenza viruses (SIVs) are influenza A viruses and they = belong to=20 the family Orthomyxoviridae. These viruses also occur in wild birds, = poultry,=20 horses and humans, but interspecies transmission is a rare event. All = influenza=20 A viruses have the same basic structure, but there are considerable = differences=20 between influenza viruses of different species and between different = =E2=80=9Csubtypes=E2=80=9D=20 that may co-circulate in one species. The surface of an influenza virus = particle=20 is covered by two types of glycoproteins: haemagglutinin (HA) and = neuraminidase=20 (NA). HA and NA are important because they elicit an antibody response = to the=20 influenza virus. Fifteen different HAs (H1 to H15) and 9 different NAs = (N1 to=20 N9) have been recognized by virologists and these form the basis for=20 classification of influenza viruses into =E2=80=9Csubtypes=E2=80=9D. =
Pigs are notable among=20 mammalian influenza virus hosts in that they harbour three different = subtypes:=20 H1N1, H1N2 and H3N2. New influenza virus subtypes can emerge in two = different=20 ways and this is referred to as antigenic =E2=80=9Cshift=E2=80=9D. One = possible mechanism is=20 interspecies transmission. The H1N1 subtype circulating in the European = swine=20 population, for example, is originally an avian virus that has spread = from wild=20 waterfowl into the pig population in 1979. A second possibility is = genetic=20 =E2=80=9Creassortment=E2=80=9D. This can occur when two different = influenza viruses=20 simultaneously infect the same host. The genome of an influenza virus is = =E2=80=9Csegmented=E2=80=9D =E2=80=93 it comprises eight single-stranded = negative-sense RNA=E2=80=99s - and the=20 8 gene segments of one virus can then mix and match with that of the = other virus=20 to form a new combination virus. Both the H3N2 and the H1N2 SIVs = circulating in=20 Europe are reassortants. The H3N2 virus is human in origin and = it=E2=80=99s HA and NA=20 resemble those of the human virus that caused the =E2=80=9CHong Kong = flu=E2=80=9D pandemic in=20 1968. This virus has later reassorted with the = =E2=80=9Cavian-like=E2=80=9D swine H1N1 virus,=20 from which it derived the internal proteins. The H1N2 virus was first = isolated=20 in Great Britain in 1994 and has a very complex history of origin. The = virus=20 appears to be a combination of the HA of a human H1N1 virus from the = early=20 1980=E2=80=99s and the NA of the swine H3N2 virus. The internal = proteins, on the other=20 hand, are =E2=80=9Cavian-like=E2=80=9D as in the swine H1N1 and H3N2 = viruses. The European H1N2=20 virus, therefore, is remarkably distinct from the H1N2 virus that has = been=20 previously reported in Japan and from H1N2 in the US.
Smaller = antigenic=20 changes in the HA or NA of an already existing influenza virus subtype = are=20 called antigenic =E2=80=9Cdrift=E2=80=9D. Generally spoken, the immunity = that develops after=20 infection with a given influenza virus subtype protects only partially = against a=20 =E2=80=9C=E2=80=99drift variant=E2=80=9D and not at all against a new = subtype. Antigenic drift is less=20 common with swine than with human influenza viruses. Still, genetic = reassortment=20 of influenza viruses appears to occur more frequently in pigs than in = any other=20 influenza virus host. This can be explained by the fact that pigs are=20 susceptible to infection with a variety of influenza A virus strains, = both of=20 avian and human origin. In addition, pigs have regular close contacts = with=20 humans and birds.

Three SIV subtypes circulate = concurrently in=20 Europe
SIV spreads readily by the air and by contact with=20 respiratory secretions from infected pigs. It is therefore impossible to = prevent=20 SIV infections by sanitary measures alone, and the virus is enzootic in = most=20 densely swine populated regions of Europe. Table 1 shows the = seroprevalence of=20 H1N1, H3N2 and H1N2 SIVs in unvaccinated sows in Flanders, Belgium, = between 2001=20 and 2003. In this region, only one out of 100 farms sampled was = completely=20 negative for any SIV subtype. Most individual sows had antibodies to a=20 combination of two (48%) or to all three (31%) subtypes, indicating = infection=20 with multiple subtypes during their lifetime. A similar situation has = been=20 reported in Germany, Italy and Spain, where the three subtypes are also=20 widespread.
Most pigs are thus born from immune sows and will have=20 maternally-derived antibodies during their first 7 to 8 weeks of life. = If pigs=20 become exposed to the influenza virus in the presence of maternal = antibodies,=20 they may be (partially) protected against the infection and they are = less likely=20 to develop disease. Most SIV infections, therefore, occur after 10 weeks = of age.=20 By the end of the fattening period, however, pigs rarely test negative = for SIV=20 antibodies. In recent serologic examinations at the slaughterhouse in = Flanders,=20 only 9% of the 168 pigs examined were SIV negative and 44% had = antibodies to two=20 different SIV subtypes.

3DTabel1=20 =

The disease =
SIV is one=20 of the rare primary respiratory pathogens of swine. This means that the = virus=20 can induce disease and lung lesions on its own. Typical swine = =E2=80=9Cflu=E2=80=9D outbreaks=20 are characterized by a rapid onset of high fever, dullness, loss of = appetite,=20 labored abdominal breathing and coughing. Weight loss can be = considerable, but=20 mortality is low and recovery occurs within 7-10 days. In studies of = acute=20 respiratory disease outbreaks on pig farms in the Netherlands in = 1996-1998, SIV=20 was diagnosed in nearly 50% of the cases. All three virus subtypes have = been=20 associated with disease and there are no indications for differences in=20 virulence between subtypes or strains. However, the infection is much = more=20 frequent than the disease. Subclinical infections are very common and = many pigs=20 become infected with the virus without ever showing clinical signs. = Another=20 possibility is that the infection with SIV does not induce the typical = flu=20 symptoms, but that it interacts with other respiratory pathogens to = induce=20 multifactorial disease. Mycoplasma hyopneumoniae, porcine reproductive = and=20 respiratory syndrome virus (PRRSV), P. multocida, H. parasuis, S. suis = and B.=20 bronchiseptica have been found in association with SIV and SIV is = considered a=20 contributor to the so-called porcine respiratory disease complex (PRDC). = Also,=20 secondary infections with bacteria may cause more severe and prolonged = disease=20 and even mortality in typical clinical cases of SI.=20

Understanding disease mechanisms
From the=20 pathogenetic viewpoint, SIV is the typical example of an acute = respiratory tract=20 infection. The virus replicates in epithelial cells of the entire = respiratory=20 tract, from the nose to the deeper lungs, but almost never enters other = tissues.=20 Immunofluorescence and immunohistochemical studies have shown that = bronchiolar=20 and alveolar epithelial cells are the major target cells (Fig. 1). = Epithelial=20 cell necrosis and an influx of neutrophils into the lung accompany the = typical=20 respiratory disease. These inflammatory cells cause obstruction of the = airways=20 and substantial lung damage by release of their enzymes. Both the = infection and=20 disease are very transient. Virus excretion in nasal swabs and virus = replication=20 in the lungs last for 6-7 days at most.
Why does not every infection = with=20 SIV result in the typical clinical symptoms? Based on our experiments, = we=20 believe that disease severity relates to the amount of virus produced in = the=20 lungs and the resulting release of inflammatory mediators by the host. = Under=20 experimental conditions, the infection can be easily reproduced, but the = typical=20 disease and lung pathology only result when pigs are inoculated with = high virus=20 doses directly into the trachea. In such intratracheal infection = studies, we=20 found massive virus titers in the lungs and high levels of several = cytokines, or=20 =E2=80=9Csignal molecules=E2=80=9D, in lung lavage fluids. The cytokines = included=20 interferon-alpha (IFN-=EF=81=A1), tumor=20 necrosis factor-alpha (TNF-=EF=81=A1),=20 interleukin-1 (IL-1) and IL-6. These cytokines are known to cause lung=20 inflammation, functional lung disturbances, fever, malaise and loss of = appetite,=20 and they can strongly enhance each other=E2=80=99s effects. These = symptoms were seen=20 within 24 hours after intratracheal inoculation of pigs with SIV and = they were=20 associated with the peak of virus replication and peak cytokine levels. = In=20 contrast, inoculation by the less invasive intranasal or aerosol = inoculation=20 routes resulted in lower virus titers in the lungs. These infections = remained=20 clinically mild or subclinical and failed to induce the massive = production of=20 cytokines in the lungs (Fig. 2). It is noteworthy that intratracheal = infection=20 studies with other respiratory viruses, such as the porcine respiratory = corona=20 virus (PRCV) or PRRS virus, also failed to induce high levels of = multiple=20 cytokines as well as obvious respiratory disease.
Together, these = data=20 support that a massive SIV replication in the lungs is required to = induce high=20 cytokine levels and the associated disease. Any factors (partial active = or=20 passive immunity, sanitary measures=E2=80=A6) likely to reduce the = extent of virus=20 replication are thus likely to prevent disease.

3DFig1=20 =

Fig. 1. Lungs of a pig = 24 hours=20 after experimental infection with SIV. (A) Immunofluorescence staining = of=20 bronchiolar epithelium, (B) Neutrophil infiltration in the bronchiolar = lumen,=20 (C) Neutrophils are the predominant cells in lung lavage fluids.=20

Effectiveness of the current SI vaccines=20
Commercial, inactivated SIV vaccines for intramuscular = administration have=20 been in use in Europe since the early 1980s. They are based on whole = inactivated=20 influenza virus, or its immunogenic proteins, HA and NA, and an oil = adjuvant.=20 Most vaccines used in Belgium and in the Netherlands contain the human = New=20 Jersey/76 (H1N1) and Port Chalmers/73 (H3N2) strains, but none of the = vaccines=20 contains an H1N2 component. The first vaccination, usually around the = age of 10=20 weeks, should consist of two injections 3 to 4 weeks apart. In sows, = bi-annual=20 revaccinations are recommended. These inactivated vaccines rely largely = on=20 circulating antibodies to the viral HA for protection. There appears to = be some=20 diffusion of these antibodies from the circulation into the lungs, where = they=20 can neutralize the virus in case of an infection. Consequently, there is = a tight=20 correlation between HI antibody titers induced by the vaccine and = protection.=20
Of all vaccines against respiratory viruses of pigs, SI vaccines are = among=20 the most effective. Under experimental conditions, a double vaccination = of SIV=20 seronegative pigs can provide complete protection against a severe = intratracheal=20 challenge with a high dose of H1N1 or H3N2 SIV. In our experiments, pigs = that=20 were not completely protected against the infection show a highly = significant=20 reduction of the challenge virus titer in their lungs when compared to=20 unvaccinated control pigs. Cytokine titers in the lungs of these = vaccinated pigs=20 were also 10 to 100 times lower than in the control pigs, and all = vaccinated=20 pigs were completely protected against disease. This led us to conclude = that=20 even a minimal reduction of the influenza virus titer in the lungs of = vaccinated=20 pigs strongly reduces cytokine levels and that this reduction is = sufficient to=20 prevent the typical disease.
Another important point is that the = commercial=20 vaccines, which contain older H1N1 and H3N2 strains, do still adequately = protect=20 against the H1N1 and H3N2 strains that are currently circulating in = swine. There=20 has been much debate as to whether antigenic drift of H1N1 and H3N2 SIVs = would=20 require replacement of the vaccine strains by more recent ones, as = occurs with=20 human and equine flu vaccines. Our and other experimental studies have=20 convincingly shown that such a replacement is not needed. One weakness = of the=20 current vaccines, however, is that they did not protect against = infection or=20 disease by the H1N2 virus in our experiments. This can be explained by = the=20 dramatic antigenic difference between the H1 of H1N1 and H1N2 viruses. = As such,=20 the antibodies induced by the H1N1 vaccine strain cannot neutralize the = H1N2=20 virus and therefore not prevent the infection. Thus, some consideration = should=20 be given to the inclusion of an H1N2 strain in SIV vaccines for use in = pigs in=20 Europe.
In the field, the efficacy of SI vaccination will depend on = numerous=20 factors. Maternal antibodies, for example, may interfere with effective=20 vaccination of piglets. This is one reason why SI vaccines are mainly = used in=20 sows, but in most European countries less than one quarter of the sow = population=20 is vaccinated. Though there is little published efficacy data on SI = vaccination,=20 serologic investigations have shown significantly higher H1N1 and H3N2 = HI=20 antibody titers, up to 640-2560, in vaccinated than in unvaccinated = sows. Sow=20 vaccination clearly results in higher maternal antibody levels in the = young=20 piglets, which may persist until the age of 12-14 weeks. It will control = disease=20 in suckling pigs and seems to protect pigs throughout the nursery phase. =

3DFig2=20 =

Fig. 2. SIV titers = in the=20 pig lung, clinical outcome and cytokine levels (log10 values) in lung = lavage=20 fluids in 3 different experimental situations: (1) intratracheal = inoculation of=20 SIV seronegative pigs, (2) intranasal inoculation of SIV seronegative = pigs, (3)=20 intratracheal inoculation of previously vaccinated pigs. The results = suggest=20 that any measures that reduce the infection pressure and/or the viral = load in=20 the lungs will reduce cytokine production and the resulting symptoms.=20

Some cross-protection between subtypes after infection = with SIV=20
Apart from protection following vaccination, we have also = studied=20 the protective immune response after a true infection with SIV. We have = focused=20 on the question as to whether pigs with infection-derived immunity to = H1N1 or=20 H3N2 are still fully susceptible to the antigenically different H1N2 = virus. To=20 examine this, several groups of pigs were inoculated intranasally with = two=20 (H1N1-H1N2, H3N2-H1N2) or three subtypes (H1N1-H3N2-H1N2) one month = apart. H1N2=20 challenge virus titers in nasal swabs were determined to evaluate = protection=20 against challenge. Serum HI antibody titers to each of the three = subtypes were=20 also determined at several time points. These data were used to gain = insights in=20 the interpretation of serological profiles from the field.
Prior = infection=20 with H1N1 or H3N2 did not protect against infection with the H1N2 virus, = while=20 H1N2 infection-immune pigs were fully protected. Still, H1N2 virus = excretion was=20 up to two days shorter in H1N1 or H3N2 immune pigs than in fully = na=C3=AFve pigs.=20 This points towards a very limited and partial cross-protection between = two=20 different SIV subtypes. A surprisingly solid cross-protection to the = H1N2 virus=20 was found in pigs with infection-immunity to both H1N1 and H3N2. The = H1N2 virus=20 was not or barely detectable in nasal swabs as well as in lung tissue of = these=20 pigs. Following intratracheal H1N2 challenge, the pigs did not show any = clinical=20 signs. Despite the protection against the H1N2 subtype, these pigs only = had=20 antibodies to H1N1 and H3N2 at the time of the H1N2 challenge. There was = thus no=20 serological cross-reaction between the subtypes in the HI test. =
Thus, though=20 cross-protection between subtypes does not occur after vaccination, it = may be=20 induced by exposure to infectious SIV. This suggests that viral proteins = other=20 than the HA, which is the major immunogenic protein in the vaccines, may = have=20 some role. A mucosal or cell-mediated immune response, which is only = stimulated=20 by infection, is probably involved.
The exact significance of these=20 experimental findings for the field situation is not yet clear. It is = possible=20 that this =E2=80=9Cheterosubtypic=E2=80=9D protection contributes to the = mild clinical course of=20 many SIV infections in the field.

Diagnosis =
The=20 diagnosis of SIV is relatively straightforward, but the timing of sample = selection is a critical issue. Because SIV replicates in the respiratory = tract=20 for only 5 to 7 days after infection, samples should be collected from = febrile,=20 acutely affected pigs. Both lung tissue or nasal swabs can be used to = isolate=20 the virus. In the diagnostic laboratory, embryonated chicken eggs or = cell=20 cultures are used for virus isolation. Virus isolation remains among the = most=20 sensitive detection methods, but it takes at least several days and up = to 1-2=20 weeks to determine the SIV subtype. The fluorescent antibody test on = frozen=20 sections of lung is a more rapid (2-3 hours) alternative. Because only = small=20 portions (4 mm sections) of lung are examined in this test, it is less = sensitive=20 than virus isolation. A commercial membrane immunoassay (Directigen = TM, Becton Dickinson) to demonstrate = influenza=20 virus in nasal swabs is also available. This test was designed to detect = influenza viruses in throat and nasal swabs of humans, but is works = equally well=20 for any influenza A virus from other species, including swine, poultry = and=20 horses. Speed is the greatest advantage of this test because it takes = only 20=20 minutes, but it is not subtype-specific.
The haemagglutination = inhibition=20 (HI) test is the most widely used serologic test for SIV. HI antibody = titres in=20 serum peak by 2-3 weeks after an infection and they begin to decline by = 3-6=20 months. Paired sera, collected at the time of the presumed SIV outbreak = (acute=20 serum) and approximately 3 weeks later (convalescent serum), are needed = for the=20 serologic diagnosis of SIV. If there has been a recent infection with a = given=20 SIV subtype, the convalescent sera will show rising antibody titers to = that=20 subtype. The HI test is highly subtype-specific and separate tests must = be=20 performed with H1N1, H3N2 and H1N2 strains as antigens. Furthermore, the = test is=20 most sensitive if these strains resemble the current field strains. =
Our pig=20 infection experiments with multiple SIV subtypes have confirmed the lack = of=20 serologic cross-reaction between subtypes. HI antibodies to a given SIV = subtype=20 generally point towards a previous infection with that subtype. On the = other=20 hand, the interpretation of HI antibody profiles may become much more = complex if=20 pigs have been subsequently exposed to different SIV subtypes. To = illustrate,=20 pigs that become infected with H1N1 followed by H1N2 or vice versa, may = show a=20 serologic booster to the first infecting subtype after the second = infection. The=20 other way round, seroconversion to a given SIV subtype can be less = pronounced if=20 pigs are partially protected against that subtype.

SIV: = a=20 zoonotic risk?
As mentioned higher, influenza viruses are=20 species-specific and this is also true for swine influenza viruses. = There are=20 only a few documented cases of transmission of SIVs to humans, often = children.=20 In these rare cases, there was no further transmission of SIV between = humans.=20 For a still unknown reason, SIVs do not manage to spread efficiently in = the=20 human population. The single exception here was the so-called = =E2=80=9CNew Jersey=E2=80=9D=20 incident in the US in 1976, during which some 500 humans became infected = with a=20 swine H1N1 influenza virus. Still, most of these infections were = subclinical and=20 there was no real swine flu pandemic. Recent serologic investigations = have shown=20 higher levels of seropositivity to H1N1 SIV in people who had close = contact with=20 pigs than in urban residents. However, these data must be interpreted = with=20 caution, because it is difficult to distinguish between antibodies to = human and=20 swine influenza viruses by the classical serologic methods.
One = difference=20 between swine and other mammalian influenza virus hosts is that swine = are=20 clearly more susceptible to infection with avian influenza viruses. In = the past,=20 it was assumed that spread of avian influenza viruses into the human = population=20 can only occur after passage or reassortment of these viruses in the = pig, but=20 this hypothesis had to be rejected recently. Indeed, some of the = so-called=20 =E2=80=9Chighly pathogenic=E2=80=9D avian influenza viruses may = occasionally transmit directly=20 to humans. Recent examples are the avian H5N1 virus in Hong Kong in 1997 = and in=20 Vietnam and Thailandin 2004-05, as well as the H7N7 virus in The = Netherlands in=20 2003. Interestingly, the H7N7 virus was also found in a limited number = of swine=20 at the time of the avian flu outbreak, and the H5N1 virus had been = occasionally=20 detected in pigs in China in 2001 and 2003. However, transmission of = such=20 viruses between pigs appears to be minimal and both viruses disappeared = from the=20 swine population. Most important, both swine and humans appear to have = been=20 infected directly by poultry, by close contact with infected chickens. = It=20 remains uncertain, therefore, whether swine can play a role in the = transmission=20 of avian influenza viruses to humans and this question definitely merits = more=20 research.

Conclusions
The = establishment of=20 the H1N2 virus in the European swine population has added an extra level = of=20 complexity to the control and serologic diagnosis of SI in Europe. To = afford=20 maximum protection, SI vaccines may need to contain all three SIV = subtypes. On=20 the other hand, some cross-subtype protection seems to occur following = natural=20 SIV infections. The presence of a third SIV subtype is thus not = necessarily=20 detrimental. Finally, continuous surveillance of the swine population = for=20 influenza viruses remains essential, because the epidemiological = situation can=20 change rapidly.

Selected references
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BROWN I.H.: The epidemiology and evolution of influenza = viruses=20 in pigs. Vet. Microbiol., 2000, 74, 29-46. =
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=C2=A9 2007 Dirk Demeulenaere Contact=20 Me