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Neutralizing antibodies against influenza A in pigs before and after the 2009 pandemic, Luxembourg
Veterinary Microbiology 169 (2014) 96–101
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short Communication
Neutralizing antibodies against influenza A in pigs before and after the 2009 pandemic, Luxembourg Nina Lutteke a, Aure´lie Sausy a, Antony P. Black a, Fe´lix Wildschutz b, Claude P. Muller a,* a Institute of Immunology, Centre de Recherche Public de la Sante´/Laboratoire National de Sante´, 20A rue Auguste Lumie`re, L-1950 Luxembourg, Luxembourg b Ministe`re de l’Agriculture, de la Viticulture et du De´veloppement rural, Administration des Services ve´te´rinaires, Division de l’Inspection ve´te´rinaire, Re´sidence St Louis 211, route d’Esch, L-1471 Luxembourg, Luxembourg
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 August 2013 Received in revised form 11 December 2013 Accepted 15 December 2013
Neutralizing antibodies against different swine influenza A viruses and pandemic H1N1 were analyzed in pigs before and after the pandemic. While in 2009 neutralization of the pandemic virus could be explained by cross-reaction with swine influenza viruses this was not the case in at least 2 farms in 2012. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Influenza A Pig Neutralizing antibodies Pandemic 2009 Cross-reaction
1. Introduction Three distinct IAV subtypes dominate currently in pigs worldwide: H1N1, H1N2 and H3N2 (Brown, 2000). In Europe, the classical H1N1 swine influenza virus (SIV), entirely of avian origin, was introduced in the swine population in 1979 (Pensaert et al., 1981) and is common in most countries throughout the continent (Van Reeth et al., 2008). H1N2 is found in European pigs since 1994; the HA component is of human origin, while the N gene originated from a swine H3N2 virus. The H3N2, a reassortant of human and swine H1N1 viruses, was introduced in 1984 and is also common in European pigs (Brown et al., 1998). The prevalence of the three virus subtypes varies between countries (Van Reeth et al., 2008). Two different H1N1 subtypes occur in pigs in the Americas: the classical SIV circulating since 1918, and a
* Corresponding author. Tel.: +352 26970620; fax: +352 490686. E-mail address: [email protected] (C.P. Muller). 0378-1135/$ – see front matter ß 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.12.009
triple reassortant with the HA and NA from the classical SIV, first detected in 1998 (Olsen, 2002). In March 2009, a new H1N1 reassortant virus with hemagglutinin (HA) properties of the American SIV emerged in Mexico, and rapidly developed into a pandemic, partially replacing seasonal influenza A strains (IAVs) (World Health Organisation, 2009). While the pandemic H1N1/2009 (pdm/09) influenza virus was efficiently transmitted from human-to-human it also preserved its tropism for swine. Since its pandemic spread, the virus was detected in pigs in countries worldwide and on all major continents (Nelson et al., 2012), suggesting frequent independent human-to-swine transmission events. Infection of pigs with pdm/09 in Europe was first reported by the end of November 2009 from Finland (OIE, 2009). The virus has also been found in pigs in countries bordering with Luxembourg (OIE, 2012). So far there is no data on IAV seroprevalence or on IAV circulating in pigs in Luxembourg. In an earlier study we investigated neutralizing antibodies against different IAVs including pdm/09, sw/B/98 and A/Luxembourg/572/2008
N. Lutteke et al. / Veterinary Microbiology 169 (2014) 96–101
in pig workers. We showed that antibodies against swine influenza were higher in pig workers than in the general population (Gerloff et al., 2011), indicating that swine influenza A virus occurs in pigs in Luxembourg. Therefore, we investigated the prevalence of antibodies against pdm/09 virus in fattening pigs from farms in Luxembourg before and after the pandemic in comparison to antibodies against other SIVs, including two European swine H1N1 viruses of avian origin (A/sw/Belgium/1/ 1998, sw/B/98; A/sw/Luxembourg/1065/2009, sw/L/09), a classical American H1N1 swine virus (A/swine/Iowa/ H04YS2/2004, sw/I/04), a European swine strain with an HA protein of human origin (A/swine/Gent/7625/1999, sw/G/99), an European swine H3N2 strain (A/swine/ Flanders/1/1998, sw/F/98) and a representative virus of the H1N1 pandemic isolated in Luxembourg (A/Luxembourg/43/2009, pdm/09). 2. The study 203 pig blood samples from 36 different farms throughout Luxembourg (up to 5 pigs/farm/time-point) were collected between June 22nd and August 6th, 2009, i.e. before sustained transmission of pdm/09 in the country
97
(Gerloff et al., 2011). In 2012, 287 blood samples were obtained from 40 of the 324 fattening pig farms in Luxembourg, between March 21st and May 14th (up to 6 pigs/farm/time-point). A representative selection of the whole pig population from throughout the country (Fig. 1) was collected at both time points from the two slaughterhouses in Luxembourg which process more than 99% of the pigs. Sera were tested by virus neutralization (VN) assay using 6 different viruses (Table 1) following the protocols of the World Health Organization (World Health Organisation, 2005). This assay was preferred over the hemagglutinin inhibition (HI) assay because the latter has been shown to be less sensitive (Rowe et al., 1999; World Health Organisation, 2005; Zhu et al., 2011). The VN assay is thought to detect both the HA1 and HA2 antibodies, the main target of neutralizing antibodies, whereas the (HI) assay detects mainly HA1 antibodies (Lim et al., 2008). Serum samples were heated to 56 8C for 30 min to inactivate complement and other unspecific inhibitors. Titers were reported as the reciprocal of the highest dilution of serum that completely neutralized viral growth. All sera were titrated in quadruplicates until a dilution of at least 1:640. For statistical analysis data was dichotomized
Fig. 1. Localization of the participating farms across Luxembourg.
N. Lutteke et al. / Veterinary Microbiology 169 (2014) 96–101
98
Table 1 Influenza A subtypes and HA origin of viruses used in this study. Virus
Short name for publication
Sub type
A/swine/Belgium/1/1998
sw/B/98
H1N1
A/swine/Luxembourg/1065/2009
sw/L/09
A/swine/Flanders/1/1998
Antigenic/genetic
Year of introduction
HA genetic characteristics
HA accession number
Complete avian
1979
FJ805962.1
H1N1
Complete avian
1979
sw/F/98
H3N2
1984
A/swine/Gent/7625/1999
sw/G/99
H1N2
A/swine/Iowa/H04YS2/2004
sw/I/04
H1N1
A/Luxembourg/43/2009
pdm/09
H1N1 pdm
Reassortant (human H3N2, swine H1N1) Reassortant (human H1N1, swine H3N2, swine H1N1) Reassortant, (classical American SIV, swine H3N2 or swine H1N2) Reassortant (European swine H1N1, American swine H1N2, American swine H3N2)
Avian-like, European Avian-like, European Human, European Human, European Swine, classical American Swine, classical American
FN423713.1
characteristics
for different cut-off points and analyzed by x2-test and for low proportions by z-test using SigmaPlot v12.5. Although, it has been estimated that neutralization titers of 80–160 provide a 50% risk reduction of infection in humans (Hancock et al., 2009), in pigs there this is no clear correlate of protection for neutralizing antibodies or a definition of a protective titer measured by virus neutralization assay. Our results, of 35–95.1% of neutralizing antibodies against any of the viruses at a cut-off of 20 (Fig. 2A and B), suggest that these may be largely unspecific. At titers of 40 or 80 essentially no reactivity was found against at least some viruses (e.g. sw/F/98 and sw/G/99) suggesting that these could be more suitable cutoffs. Because of this complication, we analyzed titers using running cut-offs for positivity starting at 40.
1994 1998
2009
HG421297 FJ842116.1 AY590823.1 GQ452235.1
68.5% of all pigs were positive for at least one virus at a cut-off 40 (31.52%, at cut-off 80) in 2009 and 53% at cutoff 40 (41.46%, 80) in 2012. In 2009, 97% of the farms had at least one pig that was positive for at least one virus at cutoff 40 (51.4%, 80). 67.6% were positive for 2 or more viruses at cut-off 40 (35.1%, 80). Similarly, in 2012, 65% of the farms had at least one pig that was positive for at least one virus at cut-off 40 (55%, 80) and in 45% positive for at least 2 or 3 viruses at cut-off 40 (37.5%, 80). As expected antibody prevalence for the two SIVs (sw/ B/98, sw/L/09) was highest both in 2009 and 2012 for all cut-offs 40 compared to the other viruses (Fig. 2A and B). In both years the seroprevalence of antibodies against sw/ B/98 and sw/L/09 was similar for most cut-offs (except 40, p 0.001 in 2009 and 80, p = 0.03 in 2012). When
Fig. 2. GMTs of neutralizing antibodies against for sw/B/98, sw/F/98, sw/G/99, pdm/09, sw/I/04, sw/L/09 in 2009 (A) and 2012 (B). Comparison of GMTs at different cutoffs for sw/B/98 (C), sw/L/09 (D), pdm/09 (E) between 2009 and 2012, *p < 0.05, **p = 0.001, ***p < 0.001. For statistical analysis data was dichotomized for different cut-off points and analyzed by x2 test. For low proportions z-test was used.
N. Lutteke et al. / Veterinary Microbiology 169 (2014) 96–101
A
R = 0.373 p = 4.06*10-5
1024
1024 256
64
64
64
16
1
pdm/09
256
4
16
1
4
16
64
256
1024
1
1024
4
1
4
16
64
256
sw/B/98
D
R = 0.538 p = 2.20*10-9
1024
1
1024
E
R = 0.512 p = 7.98*10-6
1024
64
64
64
4 1
pdm/09
256
sw/L/09
256
16 4
1
4
16
64
256
1
1024
1
4
16
64
256
1024
sw/L/09
256
16
C
R = 0.323 p = 4.48*10-3
16
4
sw/B/98
sw/B/98
B
R = 0.439 p = 1.7*10-8
256
sw/L/09
pdm/09
1024
2009
99
F
R = 0.447 p = 7.89*10-4
16 4
1
4
16
sw/I/04
64
256
1
1024
1
4
sw/I/04
16
64
256
1024
sw/I/04
2012 G
1024
256
256
64
64
16
16
1 1
4
16
64
256
1024
1024
I
R = 0.104 p = 0.230
256
4
4 1
H
R = 0.598 p = 6,65*10-13
pdm/09
R = 0.257 p = 4.39*10-3
sw/L/09
pdm/09
1024
64 16 4
1
4
sw/B/98
16
64
sw/B/98
256
1024
1
1
4
16
64
256
1024
sw/L/09
Fig. 3. Geometric mean titers of neutralizing antibodies against the different viruses. Each symbol represents the titer of one pig for 2 viruses in 2009 (A–F) and 2012 (G–I). Double negative results (strictly below 40) were excluded. Trend lines, R-values (Pearson Correlation Coefficient), p-values are shown.
comparing the two years, prevalence of antibodies against the sw/L/09 and the sw/B/98 was higher in 2012, with a significant increase for sw/B/98 for cut-offs 160 to 640 (Fig. 2C) and for sw/L/09 for cut-offs 80 to 320 (Fig. 2D). Only for sw/B/98 at cut-off 40 the opposite trend was observed (Fig. 2C). Overall the two European H1N1 SIVs and in particular sw/L/09 induced also the highest antibody titers indicating that, in Luxembourg, SIVs of ‘‘avian’’ origin are the most prevalent, and the current viruses are antigenetically more similar to the sw/L/09 virus than to the sw/B/98.
For most cut-offs 40 antibody prevalence against pdm/09 was lower than against the European SIVs in both years (except 20, 2012) (Fig. 2A and B). This was also the case for sw/I/04 in 2009. When antibody prevalences against pdm/09 where compared between the two sampling years, an increase was observed in 2012, but this was only significant for a cut-off 40 and the observed increase in antibody levels was not as strong as for the European SIVs (Fig. 2E). Interestingly pigs with anti pdm/09 positivity 80 in 2009 (n = 16) were in all cases positive (40) for at least
100
N. Lutteke et al. / Veterinary Microbiology 169 (2014) 96–101
one or two (4/16) or even three other viruses (12/16) and these were with one exception H1N1 viruses. These results are highly indicative of anti-H1 antibodies crossreacting with pandemic H1N1. This cross-reactivity was also apparent when titers of individual pigs against pdm/ 09 are directly compared to the other H1N1 viruses in particular in 2009 (R = 0.323–0.447; p = <0.00005–0.005) (Fig. 3A and C). Sw/B/98 is closely related to sw/L/09 and both represent the swine H1N1 of avian origin circulating in Europe. Cross reactivity between European swine H1N1 viruses of avian origin with the pandemic 2009 virus by HI or VN test has been shown despite distinct genetic and antigenic properties (Durrwald et al., 2010; Kyriakis et al., 2010a,b). Similarly, the HA proteins of pdm/09 and the sw/I/04 are both of the classical American swine H1N1 origin sharing genetic and antigenic properties. In 2012 these correlations were conspicuously weak (R = 0.104–0.257; p = 0.00438–0.230), especially for sw/L/ 09 (Fig. 3G and I). This can be partially explained by 2 farms with 11 pigs that had titers of 80 for pdm/09 and were negative (20) for sw/L/09 and sw/B/98. This suggests that at least the latter farm had additional contacts with H1N1 viruses, most probably with pdm/09. Their close genetic relation also explains the good correlation or cross-reactivity between the sw/L/09 and the sw/B/98 as well as the American SIV with sw/B/98 and sw/L/09 (Fig. 3B, D–F and H). Cross-reactivity between European avian H1N1 and classical North American SIV was shown (De Vleeschauwer et al., 2011; Kyriakis et al., 2010b). The higher antibody prevalence and titers in 2012 against the avian-like European H1N1 SIV shows that transmission continues in Luxembourg and may even be on the rise. First cases of pandemic H1N1 infections in humans in Luxembourg were reported by week 23 of 2009 (LNS, 2009) and first sustained human-to-human transmissions were reported by mid of September 2009 (Gerloff et al., 2011). In 2009 reactivity against pdm/09 is essentially explained by cross-reactivity as has been reported before (Gerloff et al., 2011; Krumbholz et al., 2010; Kyriakis et al., 2010b; Nelson et al., 2012; Tsukamoto et al., 2011; Wang et al., 2011), in particular when pigs are infected with several IAVs (Durrwald et al., 2010). In 2012 at least 2 farms seemed to have had contact with pdm/09. Neutralizing activity against the human-like sw/F/98 (H3N2) was weak and rare (0.99% positive at cut-off 40). Thus there was no cross-reactivity between H1N1 and the H1N2 or H3N2 viruses. HA of sw/G/99 (H1N2) is, in contrast to the other H1 viruses, of human origin and does not genetically cluster with swine-origin H1 and in pig challenge experiments there was little cross-protection between H1N2 and H1N1 or H3N2 viruses (Van Reeth et al., 2003). Similarly, significant reactivity (cut-off 80) against sw/G/99 (H1N2) was only observed in the 5 pigs in a single farm. Thus our results are in line with reports from other European countries, that the avian SIV is predominant but the H1N2 and H3N2 have not completely disappeared (OIE, 2012).
3. Conclusion Dissecting neutralizing antibodies in pigs in Luxembourg we find that avian H1N1 SIV dominates but European H1N2 and possibly H3N2 SIVs did not fully disappear and that sporadic infections by pdm/09 have occurred in about 5% of the farms. The co-circulation of these multiple IAV strains within a small geographic perimeter and even the same farms provides ample opportunities for reassortment events. Funding We thank the CRP-Sante´ (EU COST Action B28, project n8REC-LNSI-20070110) and the Ministry of Agriculture, Luxembourg for financial support. Conflict of interest The authors declare no conflicts of interest. Acknowledgments We thank Kristien van Reeth from Ghent University, Faculty of Veterinary Medicine, Laboratory of Virology, Ghent, Belgium for providing SIV strains and Albert Huberty from the Ministe`re de l’Agriculture, de la Viticulture et du De´veloppement rural, Administration des Services ve´te´rinaires, Division de l’Inspection ve´te´rinaire, Luxembourg and slaughterhouse-workers for organizing the sampling of swine blood in 2009 and 2012. We thank Anna L. Reye for providing help with ArcView 9.3.1 software. References Brown, I.H., 2000. The epidemiology and evolution of influenza viruses in pigs. Vet. Microbiol. 74, 29–46. Brown, I.H., Harris, P.A., McCauley, J.W., Alexander, D.J., 1998. Multiple genetic reassortment of avian and human influenza A viruses in European pigs, resulting in the emergence of an H1N2 virus of novel genotype. J. Gen. Virol. 79 (Pt 12) 2947–2955. De Vleeschauwer, A.R., Van Poucke, S.G., Karasin, A.I., Olsen, C.W., Van Reeth, K., 2011. Cross-protection between antigenically distinct H1N1 swine influenza viruses from Europe and North America. Influenza Other Resp. Viruses 5, 115–122. Durrwald, R., Krumbholz, A., Baumgarte, S., Schlegel, M., Vahlenkamp, T.W., Selbitz, H.J., Wutzler, P., Zell, R., 2010. Swine influenza A vaccines, pandemic (H1N1) 2009 virus, and cross-reactivity. Emerg. Infect. Dis. 16, 1029–1030. Gerloff, N.A., Kremer, J.R., Charpentier, E., Sausy, A., Olinger, C.M., Weicherding, P., Schuh, J., Van Reeth, K., Muller, C.P., 2011. Swine influenza virus antibodies in humans, western Europe, 2009. Emerg. Infect. Dis. 17, 403–411. Hancock, K., Veguilla, V., Lu, X., Zhong, W., Butler, E.N., Sun, H., Liu, F., Dong, L., DeVos, J.R., Gargiullo, P.M., Brammer, T.L., Cox, N.J., Tumpey, T.M., Katz, J.M., 2009. Cross-reactive antibody responses to the 2009 pandemic H1N1 influenza virus. N. Engl. J. Med. 361, 1945–1952. Krumbholz, A., Lange, J., Durrwald, R., Hoyer, H., Bengsch, S., Wutzler, P., Zell, R., 2010. Prevalence of antibodies to swine influenza viruses in humans with occupational exposure to pigs, Thuringia, Germany, 2008–2009. J. Med. Virol. 82, 1617–1625. Kyriakis, C.S., Gramer, M.R., Barbe, F., Van Doorsselaere, J., Van Reeth, K., 2010a. Efficacy of commercial swine influenza vaccines against challenge with a recent European H1N1 field isolate. Vet. Microbiol. 144, 67–74. Kyriakis, C.S., Olsen, C.W., Carman, S., Brown, I.H., Brookes, S.M., Doorsselaere, J.V., Reeth, K.V., 2010b. Serologic cross-reactivity with pandemic (H1N1) 2009 virus in pigs, Europe. Emerg. Infect. Dis. 16, 96–99.
N. Lutteke et al. / Veterinary Microbiology 169 (2014) 96–101 Lim, A.P., Chan, C.E., Wong, S.K., Chan, A.H., Ooi, E.E., Hanson, B.J., 2008. Neutralizing human monoclonal antibody against H5N1 influenza HA selected from a Fab-phage display library. Virol. J. 5, 130. LNS, 2009. Laboratoire National de Sante´ Grand Duche´ de Luxembourg, Surveillance sentinelle de la grippe (cited 2013). Nelson, M.I., Gramer, M.R., Vincent, A.L., Holmes, E.C., 2012. Global transmission of influenza viruses from humans to swine. J. Gen. Virol. 93, 2195–2203. OIE, 2009. World Organisation for Animal Health, WAHID Interface (cited 2013). OIE, 2012.In: World Organisation for Animal Health, Summary of the OFFLU Swine Influenza Virus Group Meeting 2012. Olsen, C.W., 2002. The emergence of novel swine influenza viruses in North America. Virus Res. 85, 199–210. Pensaert, M., Ottis, K., Vandeputte, J., Kaplan, M.M., Bachmann, P.A., 1981. Evidence for the natural transmission of influenza A virus from wild ducts to swine and its potential importance for man. Bull. World Health Organ. 59, 75–78. Rowe, T., Abernathy, R.A., Hu-Primmer, J., Thompson, W.W., Lu, X., Lim, W., Fukuda, K., Cox, N.J., Katz, J.M., 1999. Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J. Clin. Microbiol. 37, 937–943. Tsukamoto, M., Hiroi, S., Adachi, K., Kato, H., Inai, M., Konishi, I., Tanaka, M., Yamamoto, R., Sawa, M., Handharyani, E., Tsukamoto, Y., 2011.
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Antibodies against swine influenza virus neutralize the pandemic influenza virus A/H1N1. Mol. Med. Rep. 4, 209–214. Van Reeth, K., Brown, I.H., Durrwald, R., Foni, E., Labarque, G., Lenihan, P., Maldonado, J., Markowska-Daniel, I., Pensaert, M., Pospisil, Z., Koch, G., 2008. Seroprevalence of H1N1, H3N2 and H1N2 influenza viruses in pigs in seven European countries in 2002–2003. Influenza Other Resp. Viruses 2, 99–105. Van Reeth, K., Gregory, V., Hay, A., Pensaert, M., 2003. Protection against a European H1N2 swine influenza virus in pigs previously infected with H1N1 and/or H3N2 subtypes. Vaccine 21, 1375–1381. Wang, W., Anderson, C.M., De Feo, C.J., Zhuang, M., Yang, H., Vassell, R., Xie, H., Ye, Z., Scott, D., Weiss, C.D., 2011. Cross-neutralizing antibodies to pandemic 2009 H1N1 and recent seasonal H1N1 influenza A strains influenced by a mutation in hemagglutinin subunit 2. PLoS Pathog. 7, e1002081. World Health Organisation, 2005. WHO Manual on Animal Influenza Diagnosis and Surveillance. World Health Organisation, 2009. http://www.who.int/csr/disease/swineflu/updates/en/index.html. Zhu, H., Ding, X., Chen, X., Yao, P., Xu, F., Xie, R., Yang, Z., Liang, W., Zhang, Y., Li, Y., Shen, J., He, P., Guo, Z., Su, B., Sun, S., Zhu, Z., 2011. Neutralizing antibody but not hemagglutination antibody provides accurate evaluation for protective immune response to H5N1 avian influenza virus in vaccinated rabbits. Vaccine 29, 5421–5423.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short Communication
Neutralizing antibodies against influenza A in pigs before and after the 2009 pandemic, Luxembourg Nina Lutteke a, Aure´lie Sausy a, Antony P. Black a, Fe´lix Wildschutz b, Claude P. Muller a,* a Institute of Immunology, Centre de Recherche Public de la Sante´/Laboratoire National de Sante´, 20A rue Auguste Lumie`re, L-1950 Luxembourg, Luxembourg b Ministe`re de l’Agriculture, de la Viticulture et du De´veloppement rural, Administration des Services ve´te´rinaires, Division de l’Inspection ve´te´rinaire, Re´sidence St Louis 211, route d’Esch, L-1471 Luxembourg, Luxembourg
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 August 2013 Received in revised form 11 December 2013 Accepted 15 December 2013
Neutralizing antibodies against different swine influenza A viruses and pandemic H1N1 were analyzed in pigs before and after the pandemic. While in 2009 neutralization of the pandemic virus could be explained by cross-reaction with swine influenza viruses this was not the case in at least 2 farms in 2012. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Influenza A Pig Neutralizing antibodies Pandemic 2009 Cross-reaction
1. Introduction Three distinct IAV subtypes dominate currently in pigs worldwide: H1N1, H1N2 and H3N2 (Brown, 2000). In Europe, the classical H1N1 swine influenza virus (SIV), entirely of avian origin, was introduced in the swine population in 1979 (Pensaert et al., 1981) and is common in most countries throughout the continent (Van Reeth et al., 2008). H1N2 is found in European pigs since 1994; the HA component is of human origin, while the N gene originated from a swine H3N2 virus. The H3N2, a reassortant of human and swine H1N1 viruses, was introduced in 1984 and is also common in European pigs (Brown et al., 1998). The prevalence of the three virus subtypes varies between countries (Van Reeth et al., 2008). Two different H1N1 subtypes occur in pigs in the Americas: the classical SIV circulating since 1918, and a
* Corresponding author. Tel.: +352 26970620; fax: +352 490686. E-mail address: [email protected] (C.P. Muller). 0378-1135/$ – see front matter ß 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.12.009
triple reassortant with the HA and NA from the classical SIV, first detected in 1998 (Olsen, 2002). In March 2009, a new H1N1 reassortant virus with hemagglutinin (HA) properties of the American SIV emerged in Mexico, and rapidly developed into a pandemic, partially replacing seasonal influenza A strains (IAVs) (World Health Organisation, 2009). While the pandemic H1N1/2009 (pdm/09) influenza virus was efficiently transmitted from human-to-human it also preserved its tropism for swine. Since its pandemic spread, the virus was detected in pigs in countries worldwide and on all major continents (Nelson et al., 2012), suggesting frequent independent human-to-swine transmission events. Infection of pigs with pdm/09 in Europe was first reported by the end of November 2009 from Finland (OIE, 2009). The virus has also been found in pigs in countries bordering with Luxembourg (OIE, 2012). So far there is no data on IAV seroprevalence or on IAV circulating in pigs in Luxembourg. In an earlier study we investigated neutralizing antibodies against different IAVs including pdm/09, sw/B/98 and A/Luxembourg/572/2008
N. Lutteke et al. / Veterinary Microbiology 169 (2014) 96–101
in pig workers. We showed that antibodies against swine influenza were higher in pig workers than in the general population (Gerloff et al., 2011), indicating that swine influenza A virus occurs in pigs in Luxembourg. Therefore, we investigated the prevalence of antibodies against pdm/09 virus in fattening pigs from farms in Luxembourg before and after the pandemic in comparison to antibodies against other SIVs, including two European swine H1N1 viruses of avian origin (A/sw/Belgium/1/ 1998, sw/B/98; A/sw/Luxembourg/1065/2009, sw/L/09), a classical American H1N1 swine virus (A/swine/Iowa/ H04YS2/2004, sw/I/04), a European swine strain with an HA protein of human origin (A/swine/Gent/7625/1999, sw/G/99), an European swine H3N2 strain (A/swine/ Flanders/1/1998, sw/F/98) and a representative virus of the H1N1 pandemic isolated in Luxembourg (A/Luxembourg/43/2009, pdm/09). 2. The study 203 pig blood samples from 36 different farms throughout Luxembourg (up to 5 pigs/farm/time-point) were collected between June 22nd and August 6th, 2009, i.e. before sustained transmission of pdm/09 in the country
97
(Gerloff et al., 2011). In 2012, 287 blood samples were obtained from 40 of the 324 fattening pig farms in Luxembourg, between March 21st and May 14th (up to 6 pigs/farm/time-point). A representative selection of the whole pig population from throughout the country (Fig. 1) was collected at both time points from the two slaughterhouses in Luxembourg which process more than 99% of the pigs. Sera were tested by virus neutralization (VN) assay using 6 different viruses (Table 1) following the protocols of the World Health Organization (World Health Organisation, 2005). This assay was preferred over the hemagglutinin inhibition (HI) assay because the latter has been shown to be less sensitive (Rowe et al., 1999; World Health Organisation, 2005; Zhu et al., 2011). The VN assay is thought to detect both the HA1 and HA2 antibodies, the main target of neutralizing antibodies, whereas the (HI) assay detects mainly HA1 antibodies (Lim et al., 2008). Serum samples were heated to 56 8C for 30 min to inactivate complement and other unspecific inhibitors. Titers were reported as the reciprocal of the highest dilution of serum that completely neutralized viral growth. All sera were titrated in quadruplicates until a dilution of at least 1:640. For statistical analysis data was dichotomized
Fig. 1. Localization of the participating farms across Luxembourg.
N. Lutteke et al. / Veterinary Microbiology 169 (2014) 96–101
98
Table 1 Influenza A subtypes and HA origin of viruses used in this study. Virus
Short name for publication
Sub type
A/swine/Belgium/1/1998
sw/B/98
H1N1
A/swine/Luxembourg/1065/2009
sw/L/09
A/swine/Flanders/1/1998
Antigenic/genetic
Year of introduction
HA genetic characteristics
HA accession number
Complete avian
1979
FJ805962.1
H1N1
Complete avian
1979
sw/F/98
H3N2
1984
A/swine/Gent/7625/1999
sw/G/99
H1N2
A/swine/Iowa/H04YS2/2004
sw/I/04
H1N1
A/Luxembourg/43/2009
pdm/09
H1N1 pdm
Reassortant (human H3N2, swine H1N1) Reassortant (human H1N1, swine H3N2, swine H1N1) Reassortant, (classical American SIV, swine H3N2 or swine H1N2) Reassortant (European swine H1N1, American swine H1N2, American swine H3N2)
Avian-like, European Avian-like, European Human, European Human, European Swine, classical American Swine, classical American
FN423713.1
characteristics
for different cut-off points and analyzed by x2-test and for low proportions by z-test using SigmaPlot v12.5. Although, it has been estimated that neutralization titers of 80–160 provide a 50% risk reduction of infection in humans (Hancock et al., 2009), in pigs there this is no clear correlate of protection for neutralizing antibodies or a definition of a protective titer measured by virus neutralization assay. Our results, of 35–95.1% of neutralizing antibodies against any of the viruses at a cut-off of 20 (Fig. 2A and B), suggest that these may be largely unspecific. At titers of 40 or 80 essentially no reactivity was found against at least some viruses (e.g. sw/F/98 and sw/G/99) suggesting that these could be more suitable cutoffs. Because of this complication, we analyzed titers using running cut-offs for positivity starting at 40.
1994 1998
2009
HG421297 FJ842116.1 AY590823.1 GQ452235.1
68.5% of all pigs were positive for at least one virus at a cut-off 40 (31.52%, at cut-off 80) in 2009 and 53% at cutoff 40 (41.46%, 80) in 2012. In 2009, 97% of the farms had at least one pig that was positive for at least one virus at cutoff 40 (51.4%, 80). 67.6% were positive for 2 or more viruses at cut-off 40 (35.1%, 80). Similarly, in 2012, 65% of the farms had at least one pig that was positive for at least one virus at cut-off 40 (55%, 80) and in 45% positive for at least 2 or 3 viruses at cut-off 40 (37.5%, 80). As expected antibody prevalence for the two SIVs (sw/ B/98, sw/L/09) was highest both in 2009 and 2012 for all cut-offs 40 compared to the other viruses (Fig. 2A and B). In both years the seroprevalence of antibodies against sw/ B/98 and sw/L/09 was similar for most cut-offs (except 40, p 0.001 in 2009 and 80, p = 0.03 in 2012). When
Fig. 2. GMTs of neutralizing antibodies against for sw/B/98, sw/F/98, sw/G/99, pdm/09, sw/I/04, sw/L/09 in 2009 (A) and 2012 (B). Comparison of GMTs at different cutoffs for sw/B/98 (C), sw/L/09 (D), pdm/09 (E) between 2009 and 2012, *p < 0.05, **p = 0.001, ***p < 0.001. For statistical analysis data was dichotomized for different cut-off points and analyzed by x2 test. For low proportions z-test was used.
N. Lutteke et al. / Veterinary Microbiology 169 (2014) 96–101
A
R = 0.373 p = 4.06*10-5
1024
1024 256
64
64
64
16
1
pdm/09
256
4
16
1
4
16
64
256
1024
1
1024
4
1
4
16
64
256
sw/B/98
D
R = 0.538 p = 2.20*10-9
1024
1
1024
E
R = 0.512 p = 7.98*10-6
1024
64
64
64
4 1
pdm/09
256
sw/L/09
256
16 4
1
4
16
64
256
1
1024
1
4
16
64
256
1024
sw/L/09
256
16
C
R = 0.323 p = 4.48*10-3
16
4
sw/B/98
sw/B/98
B
R = 0.439 p = 1.7*10-8
256
sw/L/09
pdm/09
1024
2009
99
F
R = 0.447 p = 7.89*10-4
16 4
1
4
16
sw/I/04
64
256
1
1024
1
4
sw/I/04
16
64
256
1024
sw/I/04
2012 G
1024
256
256
64
64
16
16
1 1
4
16
64
256
1024
1024
I
R = 0.104 p = 0.230
256
4
4 1
H
R = 0.598 p = 6,65*10-13
pdm/09
R = 0.257 p = 4.39*10-3
sw/L/09
pdm/09
1024
64 16 4
1
4
sw/B/98
16
64
sw/B/98
256
1024
1
1
4
16
64
256
1024
sw/L/09
Fig. 3. Geometric mean titers of neutralizing antibodies against the different viruses. Each symbol represents the titer of one pig for 2 viruses in 2009 (A–F) and 2012 (G–I). Double negative results (strictly below 40) were excluded. Trend lines, R-values (Pearson Correlation Coefficient), p-values are shown.
comparing the two years, prevalence of antibodies against the sw/L/09 and the sw/B/98 was higher in 2012, with a significant increase for sw/B/98 for cut-offs 160 to 640 (Fig. 2C) and for sw/L/09 for cut-offs 80 to 320 (Fig. 2D). Only for sw/B/98 at cut-off 40 the opposite trend was observed (Fig. 2C). Overall the two European H1N1 SIVs and in particular sw/L/09 induced also the highest antibody titers indicating that, in Luxembourg, SIVs of ‘‘avian’’ origin are the most prevalent, and the current viruses are antigenetically more similar to the sw/L/09 virus than to the sw/B/98.
For most cut-offs 40 antibody prevalence against pdm/09 was lower than against the European SIVs in both years (except 20, 2012) (Fig. 2A and B). This was also the case for sw/I/04 in 2009. When antibody prevalences against pdm/09 where compared between the two sampling years, an increase was observed in 2012, but this was only significant for a cut-off 40 and the observed increase in antibody levels was not as strong as for the European SIVs (Fig. 2E). Interestingly pigs with anti pdm/09 positivity 80 in 2009 (n = 16) were in all cases positive (40) for at least
100
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one or two (4/16) or even three other viruses (12/16) and these were with one exception H1N1 viruses. These results are highly indicative of anti-H1 antibodies crossreacting with pandemic H1N1. This cross-reactivity was also apparent when titers of individual pigs against pdm/ 09 are directly compared to the other H1N1 viruses in particular in 2009 (R = 0.323–0.447; p = <0.00005–0.005) (Fig. 3A and C). Sw/B/98 is closely related to sw/L/09 and both represent the swine H1N1 of avian origin circulating in Europe. Cross reactivity between European swine H1N1 viruses of avian origin with the pandemic 2009 virus by HI or VN test has been shown despite distinct genetic and antigenic properties (Durrwald et al., 2010; Kyriakis et al., 2010a,b). Similarly, the HA proteins of pdm/09 and the sw/I/04 are both of the classical American swine H1N1 origin sharing genetic and antigenic properties. In 2012 these correlations were conspicuously weak (R = 0.104–0.257; p = 0.00438–0.230), especially for sw/L/ 09 (Fig. 3G and I). This can be partially explained by 2 farms with 11 pigs that had titers of 80 for pdm/09 and were negative (20) for sw/L/09 and sw/B/98. This suggests that at least the latter farm had additional contacts with H1N1 viruses, most probably with pdm/09. Their close genetic relation also explains the good correlation or cross-reactivity between the sw/L/09 and the sw/B/98 as well as the American SIV with sw/B/98 and sw/L/09 (Fig. 3B, D–F and H). Cross-reactivity between European avian H1N1 and classical North American SIV was shown (De Vleeschauwer et al., 2011; Kyriakis et al., 2010b). The higher antibody prevalence and titers in 2012 against the avian-like European H1N1 SIV shows that transmission continues in Luxembourg and may even be on the rise. First cases of pandemic H1N1 infections in humans in Luxembourg were reported by week 23 of 2009 (LNS, 2009) and first sustained human-to-human transmissions were reported by mid of September 2009 (Gerloff et al., 2011). In 2009 reactivity against pdm/09 is essentially explained by cross-reactivity as has been reported before (Gerloff et al., 2011; Krumbholz et al., 2010; Kyriakis et al., 2010b; Nelson et al., 2012; Tsukamoto et al., 2011; Wang et al., 2011), in particular when pigs are infected with several IAVs (Durrwald et al., 2010). In 2012 at least 2 farms seemed to have had contact with pdm/09. Neutralizing activity against the human-like sw/F/98 (H3N2) was weak and rare (0.99% positive at cut-off 40). Thus there was no cross-reactivity between H1N1 and the H1N2 or H3N2 viruses. HA of sw/G/99 (H1N2) is, in contrast to the other H1 viruses, of human origin and does not genetically cluster with swine-origin H1 and in pig challenge experiments there was little cross-protection between H1N2 and H1N1 or H3N2 viruses (Van Reeth et al., 2003). Similarly, significant reactivity (cut-off 80) against sw/G/99 (H1N2) was only observed in the 5 pigs in a single farm. Thus our results are in line with reports from other European countries, that the avian SIV is predominant but the H1N2 and H3N2 have not completely disappeared (OIE, 2012).
3. Conclusion Dissecting neutralizing antibodies in pigs in Luxembourg we find that avian H1N1 SIV dominates but European H1N2 and possibly H3N2 SIVs did not fully disappear and that sporadic infections by pdm/09 have occurred in about 5% of the farms. The co-circulation of these multiple IAV strains within a small geographic perimeter and even the same farms provides ample opportunities for reassortment events. Funding We thank the CRP-Sante´ (EU COST Action B28, project n8REC-LNSI-20070110) and the Ministry of Agriculture, Luxembourg for financial support. Conflict of interest The authors declare no conflicts of interest. Acknowledgments We thank Kristien van Reeth from Ghent University, Faculty of Veterinary Medicine, Laboratory of Virology, Ghent, Belgium for providing SIV strains and Albert Huberty from the Ministe`re de l’Agriculture, de la Viticulture et du De´veloppement rural, Administration des Services ve´te´rinaires, Division de l’Inspection ve´te´rinaire, Luxembourg and slaughterhouse-workers for organizing the sampling of swine blood in 2009 and 2012. We thank Anna L. Reye for providing help with ArcView 9.3.1 software. References Brown, I.H., 2000. The epidemiology and evolution of influenza viruses in pigs. Vet. Microbiol. 74, 29–46. Brown, I.H., Harris, P.A., McCauley, J.W., Alexander, D.J., 1998. Multiple genetic reassortment of avian and human influenza A viruses in European pigs, resulting in the emergence of an H1N2 virus of novel genotype. J. Gen. Virol. 79 (Pt 12) 2947–2955. De Vleeschauwer, A.R., Van Poucke, S.G., Karasin, A.I., Olsen, C.W., Van Reeth, K., 2011. Cross-protection between antigenically distinct H1N1 swine influenza viruses from Europe and North America. Influenza Other Resp. Viruses 5, 115–122. Durrwald, R., Krumbholz, A., Baumgarte, S., Schlegel, M., Vahlenkamp, T.W., Selbitz, H.J., Wutzler, P., Zell, R., 2010. Swine influenza A vaccines, pandemic (H1N1) 2009 virus, and cross-reactivity. Emerg. Infect. Dis. 16, 1029–1030. Gerloff, N.A., Kremer, J.R., Charpentier, E., Sausy, A., Olinger, C.M., Weicherding, P., Schuh, J., Van Reeth, K., Muller, C.P., 2011. Swine influenza virus antibodies in humans, western Europe, 2009. Emerg. Infect. Dis. 17, 403–411. Hancock, K., Veguilla, V., Lu, X., Zhong, W., Butler, E.N., Sun, H., Liu, F., Dong, L., DeVos, J.R., Gargiullo, P.M., Brammer, T.L., Cox, N.J., Tumpey, T.M., Katz, J.M., 2009. Cross-reactive antibody responses to the 2009 pandemic H1N1 influenza virus. N. Engl. J. Med. 361, 1945–1952. Krumbholz, A., Lange, J., Durrwald, R., Hoyer, H., Bengsch, S., Wutzler, P., Zell, R., 2010. Prevalence of antibodies to swine influenza viruses in humans with occupational exposure to pigs, Thuringia, Germany, 2008–2009. J. Med. Virol. 82, 1617–1625. Kyriakis, C.S., Gramer, M.R., Barbe, F., Van Doorsselaere, J., Van Reeth, K., 2010a. Efficacy of commercial swine influenza vaccines against challenge with a recent European H1N1 field isolate. Vet. Microbiol. 144, 67–74. Kyriakis, C.S., Olsen, C.W., Carman, S., Brown, I.H., Brookes, S.M., Doorsselaere, J.V., Reeth, K.V., 2010b. Serologic cross-reactivity with pandemic (H1N1) 2009 virus in pigs, Europe. Emerg. Infect. Dis. 16, 96–99.
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