﻿Serologic studies for swine influenza viruses (SIVs)
in humans with occupational exposure to swine have
been reported from the Americas but not from Europe.
We compared levels of neutralizing antibodies against 3
influenza viruses--pandemic (H1N1) 2009, an avian-like
enzootic subtype H1N1 SIV, and a 2007­08 seasonal
subtype H1N1--in 211 persons with swine contact and
224 matched controls in Luxembourg. Persons whose
profession involved contact with swine had more neutralizing
antibodies against SIV and pandemic (H1N1) 2009 virus
than did the controls. Controls also had antibodies against
these viruses although exposure to them was unlikely.
Antibodies against SIV and pandemic (H1N1) 2009 virus
correlated with each other but not with seasonal subtype
H1N1 virus. Sequential exposure to variants of seasonal
influenza (H1N1) viruses may have increased chances for
serologic cross-reactivity with antigenically distinct viruses.
Further studies are needed to determine the extent to which
serologic responses correlate with infection.
Pandemic (H1N1) 2009 influenza virus resulted from
genetic reassortment between at least 2 swine influenza
viruses (SIVs) (1). Hemagglutinin (HA) of this novel
subtype H1N1 virus is similar to that of classical swine
influenza virus and the triple reassortant subtype H1N1
viruses that are endemic in swine populations in North
America. At the time of its detection in humans, pandemic
(H1N1) 2009 virus had never been detected in swine
populations anywhere, but it is believed to have circulated
undetected in regions with little or no surveillance for
influenza viruses in swine. Because this virus has not
been reported by the European Surveillance Network for
Influenza in Pigs (www.esnip.ugent.be) since the network's
inception in 2001, it was most likely absent in swine in
western Europe. By the end of 2009, pandemic (H1N1)
2009 virus infection of swine had been reported in Norway
(2); sporadic cases have been reported in a few other
European countries (e.g., Germany, Italy, Denmark) (3).
The swine were probably infected by contact with infected
humans, whereas transmission from swine to humans has
not yet been documented. Pandemic (H1N1) 2009 virus
is the first swine-origin virus that is readily transmitted
between humans (4).
Human infections with SIVs are rare. During 1958­
2005, only 50 cases of zoonotic infections were reported;
most were in persons who had contact with swine (5).
Limited secondary transmission to close contacts has been
reported but appears to be rare, and to our knowledge,
sustained human-to-human transmission of enzootic SIVs
has never been noted (6). Some serologic studies suggest
that persons who work with swine are at increased risk for
zoonotic infection with SIVs (7­12).
The predominant subtype H1N1 SIVs in Europe were
introduced from wild ducks to swine in 1979 and have
an entirely avian-derived genome (13­15). These viruses
are designated as avian-like viruses and are antigenically
distinct from subtype H1N1 SIVs in North America and
from pandemic (H1N1) 2009 virus. Few cases of human
infection with these avian-like swine subtype H1N1 viruses
have been reported; chains of transmission have not been
found (5,9,15), and no serologic studies have provided
indirect evidence of transmission of SIVs to humans in
Europe (15).
Swine Influenza Virus Antibodies in
Humans, Western Europe, 2009
Nancy A. Gerloff, Jacques R. Kremer, Emilie Charpentier, Aurélie Sausy, Christophe M. Olinger,
Pierre Weicherding, John Schuh, Kristien Van Reeth, and Claude P. Muller
Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 17, No. 3, March 2011 403
Author affiliations: Centre de Recherche Public de la Santé/
Laboratoire National de Santé, Luxembourg, Grand-Duchy of
Luxembourg (N.A. Gerloff, J.R. Kremer, E. Charpentier, A. Sausy,
C.P. Muller); Laboratoires Réunis, Junglinster, Luxembourg
(C.M. Olinger, J. Schuh); Health Directorate, Luxembourg (P.
Weicherding); and Ghent University, Ghent, Belgium (K. Van Reeth)
DOI: 10.3201/eid1703100581
RESEARCH
Studies in the United States, United Kingdom, and
Finland found antibodies against pandemic (H1N1) 2009
virus in elderly persons (16­18). These antibodies can
be explained by antigenic evolution of seasonal human
influenza (H1N1) viruses that are derived from the 1918
pandemic virus (such as the classical swine influenza
[H1N1] virus) but have undergone greater antigenic drift
than the swine virus (19). Antigenically, the influenza
(H1N1) viruses that circulated among humans before the
1950s are probably more closely related to the classical
swine virus and thus to the pandemic (H1N1) 2009 virus
than to contemporary human subtype H1N1 viruses. We
investigated whether persons whose professions involve
contact with swine (swine workers [SWs]) have neutralizing
antibodies against 3 influenza viruses: pandemic (H1N1)
2009 virus, a European avian-like subtype H1N1 SIV, and
a 2007­08 seasonal influenza subtype H1N1 (seasonal
influenza) virus.
Methods
Study Population
During July 20­28, 2009, blood was collected from
211 healthy persons with past or present professional
contact with swine. All participants gave informed consent
and completed a questionnaire about the nature of their
swine contacts (occupation, duration, frequency), influenza
vaccination, and influenza infection history. No participant
reported having been infected with pandemic (H1N1) 2009
virus. A total of 224 control serum samples were obtained
fromtheserumbankoftheLaboratoiresReunis,Junglinster,
Luxembourg. The samples, from the general population of
Luxembourg, had been submitted in December 2008 for
routine serologic testing. Because of ethical constraints,
no further information was gathered from controls. The
study was approved by the National Ethical Committee for
Research in Humans.
Virus Neutralization Assay
According to recommended World Health
Organization protocols (20), serum samples were tested by
virus neutralization assay against an influenza A (H1N1)
virus strain isolated from a patient in Luxembourg in
July 2009 (A/Luxembourg/43/2009). Complete genome
analyses revealed that the sequence was almost identical
to the prototype vaccine virus (A/California/7/2009) and
represented a typical North American/European pandemic
(H1N1) 2009 virus (4). Nucleotide sequences are available
from GenBank (accession nos. FN423708­15). A/swine/
Belgium/1/98 is representative of the avian-like subtype
H1N1 SIVs that are enzootic in swine populations of
western Europe (21). Both viruses have an antigenically
distinct H1 and 72% aa identity in the HA1 region (93%
and 98% aa identity in neuraminidase [NA] and matrix [M]
proteins) (22). A representative of the 2007­08 seasonal
influenza virus was included in the assay and had 73% and
74% identity in HA1 proteins compared with pandemic
(H1N1) 2009 virus and SIV (A/Luxembourg/572/2008 HA
gene, accession no. FR716024).
Positive control serum was collected from 5 patients >5
weeks after recovery from a laboratory-confirmed infection
with pandemic (H1N1) 2009 virus and from a previously
unexposed pig 4 weeks after it had been experimentally
infected with A/swine/Belgium/1/98 (H1N1) (21). Before
the assay was conducted, all samples were heated to
56°C for 30 min to inactivate complement and unspecific
inhibitors. Titers were reported as the reciprocal of the
highest dilution of serum that completely neutralized virus
growth. Samples were first screened in duplicate with a
1:10 dilution. All samples that showed virus neutralization
in 1 well were further titrated in quadruplicate up to a
dilution of at least 1:320. Control samples positive for both
viruses were included in all assays.
Statistical Analyses
Geometric mean titers (GMTs) were calculated
for each person from quadruplicate serum samples. All
negative samples were given an arbitrary GMT of 5. GMTs
were compared by using the nonparametric Wilcoxon
rank-sum test. To examine bivariate risk factors associated
with antibody prevalence, we dichotomized GMTs of all
positive samples for different cutoff points (>10 to >80)
and analyzed them by 2
test and, for low proportions, by
z-test. The distribution of antibody levels was checked for
associations with multiple risk factors by using proportional
odds modeling (23,24). Statistical analyses were performed
by using SigmaStat version 3.1 (San Jose, CA, USA) and
SPSS version 18 (Chicago, IL, USA).
Results
Study Population
Mean age of the 211 SWs was 48.2 years (range
18­94 years); 67.8% were male (Table 1). Most (84.8%)
SWs reported having worked daily in close contact with
swine (distance <1 m, 83%) for >10 years (73.5%). Among
the SWs, 133 were involved in pig breeding, fattening, or
general pig farming; 51 were slaughterhouse workers; 12
were veterinarians; 13 were butchers; and 2 were hunters.
The 224 controls were matched with SWs by age and sex
(Table 1).
Antibodies against Pandemic (H1N1) 2009 Virus
GMTs of antibodies against pandemic (H1N1) 2009
virus (Table 2) were significantly higher for SWs than for
controls (p = 0.004). Table 3 shows that 2× more SWs
404 Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 17, No. 3, March 2011
Swine Influenza Virus Antibodies in Humans
than controls had neutralizing antibodies against pandemic
(H1N1) 2009 virus for the lowest cutoff value (p = 0.001).
This ratio slightly increased with rising cutoff values and
remained significant to a cutoff >160 (Table 3). In all age
groups, 2× more SWs than controls had antibodies against
pandemic (H1N1) 2009 virus (cutoff >10), except for
persons >60 years of age (Table 4). For SWs and controls
>60 years of age, GMTs for pandemic (H1N1) 2009 virus
were similar (p = 0.897; Table 2). GMTs were significantly
higher for younger than for older (>60 years) SWs (but
not controls) (Table 2). Among SWs, antibodies against
pandemic (H1N1) 2009 virus tended to decrease with
age for all cutoff values; among controls, the same was
observed for cutoffs >10 to >40. Thus, younger SWs more
often had higher levels of antibodies against pandemic
(H1N1) 2009 virus than did controls and older SWs. The
difference between SWs and controls disappeared in
older age groups and was weaker when older and younger
controls were compared.
Antibodies against SIV
Similar to findings for pandemic (H1N1) 2009 virus,
GMTs for SIV were higher among SWs than controls;
however, the difference was not significant (Table 2; p
= 0.168). More SWs than controls had positive SIV titers
regardless of the cutoff (Table 3). These differences were
significant for cutoffs >20 to >160 and increased with higher
cutoffs (Table 3). Comparable to findings for pandemic
(H1N1) 2009 virus, for age groups up to 60 years antibodies
against SIV were found in 1.2­2× more SWs than controls
(cutoff >10; Table 4); GMTs were significantly higher
among SWs than controls in this age group (Table 2; p =
0.028). Seroprevalences and GMTs were similar for persons
>60 years of age from each group (Tables 2, 4).
In contrast to findings for pandemic (H1N1) 2009
virus, the highest proportion of seropositive persons was
found in older age groups, SWs >50 and controls >60 years
(Table 4). GMTs were significantly higher among older
(>60 years) than younger controls (p = <0.001) but differed
little among SWs (Table 2; p = 0.293).
Thus, antibody titers for SIV were found more often
and were higher among SWs than controls. In contrast to
findings for pandemic (H1N1) 2009 virus, titers for SIV
were found more often and were higher for older than
younger controls; for SWs, titers were found more often
among older persons but values were similar.
Antibodies against Pandemic (H1N1) 2009
Virus and SIV
Among SWs, for all cutoff values seroprevalence
was higher for SIV than for pandemic (H1N1) 2009 virus.
The same was found for controls but only for lower titers
(10 and 20; Table 3). The differences between antibody
positivity for each of the 2 viruses increased with age among
SWs and controls (Table 4). Comparing seroprevalences for
pandemic (H1N1) 2009 virus to those for SIV, differences
were significant only for SWs >60 years (p = 0.002). Also,
significantly more controls of the same age group (>60
years) had antibodies against SIV (62.2%) than against
pandemic (H1N1) 2009 virus (6.7%, p<0.001; Table 4).
The proportion of older (>60 years) SIV-seropositive
controls (62.2%) differed significantly from the proportion
Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 17, No. 3, March 2011 405
Table 1. Characteristics of persons tested for 3 influenza viruses,
Luxembourg, 2008­2009*
Characteristic
Swine workers,
no. (%), n = 211
Controls, no.
(%), n = 224
Sex
M 143 (67.8) 151 (67.4)
F 68 (32.2) 73 (32.6)
Age group, y§
18­40 69 (32.7) 80 (35.7)
41­50 59 (28) 58 (25.9)
51­60 39 (18.5) 41 (18.3)
61­94 44 (20.9) 45 (20.1)
Profession
Farmer 133 NA
Slaughterhouse worker 51 NA
Other¶ 27 NA
Years worked with swine
<1 4 (1.9) NA
1­4 26 (12.3) NA
5­10 26 (12.3) NA
>10 155 (73.5) NA
Unknown 0 224
Frequency of swine contact
Rarely 3 (1.4) NA
Monthly 2 (0.9) NA
Weekly 25 (11.8) NA
Daily 179 (84.8) NA
Unknown 2 (0.9) NA
Frequency of close contact (<1 m) with swine
Never 1 (0.5) NA
Rarely 3 (1.4) NA
Occasionally 10 (4.7) NA
Often 22 (10.4) NA
Always 175 (82.9) NA
Self-reported influenza vaccine in past 5 y
No/unsure 155 (73.5) NA
Yes 56 (26.5) NA
Self-reported infection with seasonal influenza
No 145 (68.7) NA
Yes 57 (27.0) NA
Exposure to swine
Only until 1997 26 NA
Only until 2007 59 NA
Until time of collection 152 NA
*NA, not available.
Sampled in July 2009.
Sampled in December 2008.
§Mean (median) age 48.2 (48) years for swine workers, 47.6 (47.2) years
for controls.
¶Veterinarian, butcher, hunter.
RESEARCH
of younger (<60 years) SIV-seropositive controls (17.3%;
p<0.001).
Thus, for both groups, more persons had antibodies
against SIV than against pandemic (H1N1) 2009 virus, and
differences in positivity decreased with increasing titers.
Antibodies against SIV were more common among older
persons, and antibodies against pandemic (H1N1) 2009
virus were more common among younger persons.
Antibodies against SIV and Pandemic (H1N1)
2009 Virus
Antibody titers of convalescent-phase serum samples
from patients with pandemic (H1N1) 2009 virus were
16× higher for pandemic (H1N1) 2009 virus than for SIV
(GMTs 226.2 vs. 13.5, respectively), indicating low cross-
reactivity between these viruses. Similarly, in a pig serum
sample, GMT for SIV (>1,280) was 128× lower than that
for pandemic (H1N1) influenza (8).
406 Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 17, No. 3, March 2011
Table 2. Geometric mean titers for 3 influenza viruses in swine workers and controls, Luxembourg, 2008­2009*
Virus (strain) and participant age, y
Study sample, % (95% CI)
p value
Swine workers, n = 211 Controls, n = 224
Pandemic (H1N1) 2009 (A/Luxembourg/43/2009)
All 8.7 (7.5­10) 6.1 (5.6­6.6) 0.004
<60 9.2 (7.6­11.1) 6 (5.5­6.7) <0.05
>60 5.6 (4.5­6.9) 5.4 (4.6­6.4) 0.897
Avian-like SIV (H1N1) (A/swine/Belgium/1/98)
All 10.3 (8.8­12) 7.7 (6.9­8.5) 0.168
<60 9.8 (8.1­11.8) 6.4 (5.8­7)§ <0.05
>60 11.2 (8­15.5) 13.6 (9.9­18.5) 0.170
Seasonal influenza (H1N1) (A/Luxembourg/572/2008)¶
All 23.2 (20.3­26.4) 13.9 (12.1­15.9) <0.001
<60 21.3 (18.3­24.7) 12.4 (10.7­14.4)# <0.001
>60 30.6 (22.7­41.1) 20.1 (15.3­28.7) 0.083
*%, no. persons/total no. persons in age groups with geometric mean titer cutoff >10. SIV, swine influenza virus.
p value <0.05 for significance were calculated by using the Wilcoxon rank-sum test. Boldface indicates significance (p<0.05).
p<0.05, compared with swine contacts of the age group >60 y against pandemic (H1N1) 2009 virus.
§p<0.001, compared with controls of the age group >60 y against avian-like SIV.
¶Data for 210 swine workers, 221 controls.
#p = 0.001, compared with controls of the age group >60 y against seasonal influenza (H1N1).
Table 3. Neutralizing antibody reactivity against 3 influenza viruses in swine workers and controls, Luxembourg, 2008­2009*
Virus (strain) and cutoff value
Swine workers,
no. (%; 95% CI), n = 211
Controls,
no. (%; 95% CI), n = 224 p value
Pandemic (H1N1) 2009 (A/Luxembourg/43/2009)
 >10 46 (21.8; 16.8­27.9) 23 (10.3); 6.9­14.9) 0.001§
 >20 37 (17.5; 13­23.2) 16 (7.1; 4.4­11.3) 0.001§
 >40 31 (14.7; 10.6­20.1) 12 (5.4; 3.1­9.1) 0.002§
 >80 14 (6.6; 4­10.8) 4 (1.8; 0.7­4.5) 0.02§
 >160 6 (2.8; 1.3­6.06) 0 (0; 0­1.2) 0.033¶
 >320 5 (2.4; 1­5.4) 0 (0; 0­1.2) 0.061¶
Avian-like SIV (H1N1) (A/swine/Belgium/1/98)
 >10 66 (31.3) 25.4­37.8) 59 (26.3; 21­32.5) 0.289§
 >20 57 (27; 21.5­33.4) 38 (17; 12.6­22.4) 0.015§
 >40 39 (18.5; 13.8­24.3) 12 (5.4; 3.1­9.1) <0.001§
 >80 21 (10; 6.6­14.7) 4 (1.8; 0.7­4.5) <0.001§
 >160 9 (4.3; 2.3­7.9) 1 (0.4; 0.1­2.5) 0.019¶
 >320 4 (1.9; 0.7­4.8) 1 (0.4; 0.1­2.5) 0.331¶
Seasonal influenza (H1N1) (A/Luxembourg/572/2008)#
 >10 183 (87.1; 83­91.9) 132 (59.7; 53.2­66) <0.001§
 >20 125 (59.5; 52.8­65.9) 76 (34.4; 28.4­40.9) <0.001§
 >40 61 (29; 23.3­35.5) 39 (17.6; 13.2­23.2) 0.007§
 >80 21 (10; 6.6­14.9) 17 (7.7; 4.8­12) 0.500§
 >160 14 (6.7; 3.9­11) 7 (3.2; 1.4­6.5) 0.144§
 >320 11 (5.2; 2.9­9.2) 4 (1.8; 0.5­4.7) 0.093§
*Values are no. persons with antibodies. CI, confidence interval; SIV, swine influenza virus. Boldface indicates significance (p<0.05).
p<0.05 when compared with swine workers against avian-like SIV (H1N1) of the same titer cutoff.
p<0.003 when compared with swine workers against avian-like SIV (H1N1) of the same titer cutoff.
§2
test on 2-way table.
¶z-test.
#Data for 210 swine workers, 221 controls.
Swine Influenza Virus Antibodies in Humans
Among 66 SIV-positive serum samples from SWs,
only 28 were also positive for pandemic (H1N1) 2009
virus (GMT cutoff >10). GMTs of at least single positive
samples correlated significantly with each other (R2
=
0.5, correlation coefficient [CC] = 0.4, p<0.001; Figure,
panel C); and GMTs for SIV were significantly higher
than corresponding GMTs for pandemic (H1N1) 2009
virus (48, 95% CI 38.4­60.1, and 16.3, 95% CI 11.3­22.8,
respectively; p<0.001). To the contrary, among SIV-
positive controls GMTs for SIV did not correlate with
GMTs for pandemic (H1N1) 2009 virus (R2
<0.01, CC =
0.322; Figure, panel D).
Among SWs, being SIV positive increased the odds
of being positive for pandemic (H1N1) 2009 virus by 2.4×
(odds ratio [OR] 95% CI 1.3­4.3). Among controls, these
chances were increased by 6× (OR 95% CI 2.9­12.6).
Seasonal Influenza Virus Compared with
Pandemic (H1N1) 2009 Virus and SIV
GMTs for seasonal influenza virus were significantly
higher among SWs than controls (Table 2), and
significantly more SWs than controls had antibodies
against seasonal influenza virus, at least for titers 10 to 40
(Table 3). Among all age groups, more SWs than controls
had antibodies against seasonal influenza (Table 4). GMTs
among controls >60 years of age were significantly higher
than those among younger controls (Table 2). Significantly
more SWs and controls had antibodies against seasonal
influenza virus than against pandemic (H1N1) 2009 virus
and SIV (Table 3).
All SWs with antibodies against pandemic (H1N1)
2009 virus also had antibodies against seasonal influenza
virus with the following exceptions: 1) GMTs were
significantly higher for pandemic (H1N1) 2009 virus (50.8,
95% CI 37.7­68.4) than for seasonal influenza viruses
(31.5, 95% CI 26.6­37.4) (p = 0.001); 2) GMTs of at least
single positive serum samples did not correlate (R2
<0.01,
CC = 0.231; Figure, panel A); and 3) 17 of 21 samples
with seasonal influenza virus titers >80 were negative for
pandemic (H1N1) 2009 virus (cutoff <10). No correlation
was found between GMTs of samples positive for seasonal
influenza virus and SIV (R2
<0.01, CC = 0.339; Figure,
panel B). These results may indicate no substantial cross-
reactivity between antibodies against pandemic (H1N1)
2009 virus or SIV and at least a recent seasonal influenza
virus.
Risk Factors
Odds of having antibodies against pandemic (H1N1)
2009 virus were 2.4× (95% CI 1.4­4.2) to 3.9 (95% CI 1.3­
12) greater for SWs than for controls (cutoffs >10 to >160).
Odds of having antibodies against SIV were 1.3× (95% CI
0.8­1.9) to 9.9 (95% CI 0.5­38.9) greater for SWs than for
controls (cutoffs >10 to >80). Odds of being SIV positive
were slightly higher for farmers (OR 2.3, 95% CI 1.1­5)
than for slaughterhouse workers; odds of being positive for
pandemic (H1N1) 2009 virus were only slightly higher for
farmers than for slaughterhouse workers (OR 1.2, 95% CI
0.6­2.5). ORs for being positive for pandemic (H1N1) 2009
virus and for SIV were slightly higher for male SWs (1.7,
95% CI 0.8­3.5, and 1.1, 95% CI 0.6­2.3, respectively;
cutoff >10). Among SWs, 26.5% self-reported receiving >1
dose of seasonal influenza vaccine during the past 5 years;
among vaccinated SWs, the odds of having antibodies
against pandemic (H1N1) 2009 virus (OR 1.3, 95% CI 0.6­
2.6) as well as against SIV (OR 1.3, 95% CI 0.7­2.5; cutoff
Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 17, No. 3, March 2011 407
Table 4. Neutralizing antibody reactivity >10 for 3 influenza viruses in swine workers and controls, Luxembourg, 2008­2009*
Participant
age in 2009, y
Pandemic (H1N1) 2009
(A/Luxembourg/43/2009)
Avian-like SIV (H1N1)
(A/swine/Belgium/1/98)
Seasonal influenza (H1N1)
(A/Luxembourg/572/2008)
Swine workers Controls Swine workers Controls Swine workers Controls
<40 22/69 (31.9;
22.1­43.6)
12/80 (15;
8.8­24.4)
19/69 (27.5;
18.4­39.0)
15/80 (18.8;
11.7­28.7)
58/68 (85.3;
6.9­93.7)
50/78 (64.1;
53.5­74.8)
41­50 10/59 (16.9;
9.5­28.5)
5/58 (8.6;
3.7­18.6)
11/59 (18.6;
10.7­30.4)
8/58 (13.8;
7.2­24.9)
46/59 (78;
67.4­88.6)
29/57 (50.9;
37.9­63.9)
51­60 9/39 (15.3;
12.7­38.3)
3/41 (7.3;
2.5­19.4)
17/39 (43.6;
29.3­59.0)
8/41 (19.5;
10.2­34.0)
37/39 (94.9;
88.0­101.8)§
19/41 (46.3;
31.1­61.6)
>60 5/44 (8.6;
5.0­24.0)¶
3/45 (6.7;
2.3­17.9)#
19/44 (43.2;
29.7­57.8)
28/45 (62.2;
47.6­74.9)
42/44 (95.5;
89.3­101.6)**
34/45 (75.6;
63.0­88.1)
18­94 (total) 46/211 (21.8;
16.8­27.9)
23/224 (10.3;
6.9­14.9)
66/211 (31.3;
25.4­37.8)
59/224 (26.3;
21.0­32.5)
183/210 (87.1;
83.0­91.9)
132/221 (59.7;
53.2­66.0)
*Values are no. persons/total no. persons in age groups with antibody reactivity >10 (%; 95% confidence interval). p values <0.05 cutoff for significance
were calculated by using the 2
test. SIV, swine influenza virus.
p = 0.012, compared with controls of the same age group against pandemic (H1N1) 2009.
p = 0.037, compared with controls of the same age group against avian-like SIV (H1N1).
§p<0.001, compared with controls of the same age group against seasonal influenza (H1N1).
¶p = 0.002, compared with swine workers of the same age group against avian-like SIV (H1N1).
#p<0.001, compared with controls of the same age group against avian-like SIV (H1N1).
**p<0.05, compared with controls of the same age group against seasonal influenza (H1N1).
p<0.05, compared with swine workers against avian-like SIV (H1N1).
p<0.001, compared with controls against avian-like SIV (H1N1).
RESEARCH
>10) were slightly higher than those for unvaccinated SWs.
Odds of having antibodies against pandemic (H1N1) 2009
virus were slightly higher for SWs exposed to swine until
the time of sampling (OR 1.5, 95% CI 0.7­3.3) in 2009
than for those who had no contact with swine after 2007.
OR for having antibodies against SIV for SWs with pig
contact until time of sampling was 0.5 (95% CI 0.2­1.1)
compared with that for persons who had no contact after
1997. Thus, no significant associations were found between
year of exposure and seroprevalence of antibodies against
either virus.
Discussion
At the time of blood collection from SWs (late
July 2009), pandemic (H1N1) 2009 had spread to all
continents, but intensity was still low in Europe, especially
in Luxembourg and its neighboring countries. The only
countries in which infection rates increased were the United
Kingdom, Ireland, and Spain (where sporadic outbreaks
occurred) (25). In 2009, Luxembourg had an intensive
active surveillance system for influenza-like illnesses.
Follow-up for all patients with suspected cases included
patient travel history, RNA extraction, and PCR to detect
pandemic (H1N1) 2009 virus. All patients with confirmed
408 Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 17, No. 3, March 2011
Figure. Geometric mean titers (>10) of antibodies against pandemic (H1N1) 2009 virus, seasonal influenza (H1N1) virus, and swine
influenza virus of swine workers (A, B, C) and controls (D). Each symbol represents titer of 1 person; only persons with positive results
(>10) for at least 1 of the 2 viruses of the panel are shown. Trend lines are shown; R2
values were R2
>0.01 for panels A, B, and D and R2
= 0.5 for panel C.
Swine Influenza Virus Antibodies in Humans
disease were monitored until at least early August. Patients
and their contacts received prompt antiviral drug treatment,
and home quarantine was recommended. In Luxembourg,
60 cases were reported and confirmed around the time
that blood collection from SWs was ending. Until end
of June 2009, almost all Luxembourg patients were
epidemiologically unrelated, and the source of infection
was not determined for one fifth (26). The first sustained
transmissions were noted by mid September (J. Mossong,
pers. comm.). The first cases of pandemic (H1N1) 2009 in
swine on the European mainland were reported in January
2010 (3). Nevertheless, the difference in the time of blood
collection from controls (December 2008) and from SWs
(July 2009) is a limitation of our study.
The virus neutralization assay used measures
neutralizing antibodies mainly against HA because
antibodies were in the assay only during the virus
entry phase (20). Nevertheless, we cannot exclude that
residual antibodies against NA and M (93% and 98% aa
identity between pandemic [H1N1] 2009 virus and SIV,
respectively) may contribute to neutralization (27).
Because there is no correlate of protection for
neutralizing antibodies or a definition of a positive titer
measured by virus neutralization assay (28), we analyzed
titers by using running cutoff values for positivity and
compared GMTs. This analysis showed significantly
higher prevalence of neutralizing antibodies against
pandemic (H1N1) 2009 virus in SWs than in controls, and
seropositivity decreased with age. Younger (<60 years)
SWs had higher titers, and 2× more SWs than age-matched
controls had neutralizing antibodies against pandemic
(H1N1) 2009.
No evidence indicates that pandemic (H1N1) 2009
virus was present in swine in Europe in or before July 2009.
Reactivity with pandemic (H1N1) 2009 virus correlated
best with antibodies against SIV. Although this correlation
was highly significant among SWs with relatively high
titers for SIV, no such correlation was found among
controls, in whom antibody levels against SIV were low.
We speculate that the difference between the cohorts may
reflect cross-reactive antibodies to another influenza virus
more similar to SIV (with or without a minor contribution
of antibodies against seasonal influenza) in SWs, in contrast
to low, mainly cross-reacting seasonal influenza virus
antibodies in controls. Serologic cross-reaction between
SIV and pandemic (H1N1) 2009 virus in pigs was recently
reported (22). Our results also showed that reactivity with
pandemic (H1N1) 2009 (or SIV) in either cohort cannot
be explained by cross-reactivity with a recent seasonal
influenza virus used in this study. Nevertheless, because
more SWs than controls were exposed to seasonal influenza
virus, we cannot exclude the possibility that antibodies to
pandemic (H1N1) 2009 virus or to SIV in the SWs may be
caused by a more complex history of exposure to seasonal
influenza virus of subtype H1 or to subclinical infections
with pandemic (H1N1) 2009 virus during the first months
of the pandemic.
Our finding of low levels of neutralizing antibodies
against pandemic (H1N1) 2009 in controls (general
population) is in agreement with findings of previous
studies (29). Our findings that titers were less common
but higher for older controls contrast with reports from
the United Kingdom and Finland (16,17) but agree with
findings of 2 studies in China, where elderly persons (>60
years) had few or no neutralizing antibodies against this
virus (30,31).
Our study also showed significantly higher prevalence
of neutralizing antibodies against SIV in SWs than in the
controls at cutoffs >20 to >160, but differences in GMTs
were not significant. Similar serologic studies in humans
in the United States showed markedly elevated antibody
titers for North American SIVs of subtype H1N1 and
H1N2 in SWs compared with controls (5,8,10,11,32,33).
These studies used hemagglutination inhibition instead of
virus neutralization assays and reported ORs for increased
serologic responses instead of seroprevalence rates. The
reported ORs, however, seem to be higher than those in
our study (8,32,33) and could be partially explained by
exclusion of persons with swine exposure in the US control
groups.
Most persons undergo sequential infections with
multiple antigenic variants of human influenza subtype
H1N1 and H3N2 viruses throughout their lives. Such
infections strongly increase the odds for serologic cross-
reactions with antigenically distinct H1 viruses, as
documented in experimental studies with pigs (22), and
may explain why older persons in the general population
have higher antibody titers to SIV than their younger
counterparts. Both older and younger controls are unlikely
to have been infected with SIV, but older persons have been
exposed to a wider variety of human seasonal influenza
viruses. This exposure is also reflected by a significant
difference in GMTs for recent seasonal influenza virus
in older than younger controls. In Luxembourg, elderly
persons may have had contact with swine because during
1920­1947 in Luxembourg, 50%­22% of all households
kept >5 pigs, but before 1979, there was no apparently
substantial swine influenza activity in this part of Europe
(14). Apart from antibodies to SIV, a few controls also had
antibodies to pandemic (H1N1) 2009 virus, but these did
not correlate with each other, suggesting a different cross-
reactivity pattern than that for SWs. These findings show
that in the absence of paired serum samples, presence of
neutralizing antibodies to a given influenza virus does
not necessarily reflect infection with that virus. Elevated
antibody titers to SIV in part of the SWs may have resulted
Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 17, No. 3, March 2011 409
RESEARCH
from exposure to the virus, but further studies are required
to determine all possible causes.
In conclusion, titers of antibodies against pandemic
(H1N1) 2009 virus and against an avian-like subtype H1N1
influenza virus were found more frequently and were
higher for SWs than for controls. These titers cannot be
explained by cross-reactivity with antibodies from recent
seasonal influenza viruses. Neutralizing antibodies to both
subtype H1N1 viruses showed some degree of correlation.
Further studies are needed to determine incidence of
zoonotic SIV infections and the extent to which serologic
responses correlate with infection. Neutralizing antibodies
should confer at least partial protection against infection,
reducing the risk that the avian-like subtype H1N1 SIV will
cause major outbreaks of disease in humans.
Acknowledgments
We thank Constant Muellesch and Gerard Heitz for
assistance in recruiting study participants and Joel Mossong for
his scientific expertise.
This research was supported by the Ministry of Health.
N.A.G. received funding from the "Fonds National de la
Recherche" of the Ministry of Research of the Grand-Duchy of
Luxembourg.
Dr Gerloff is a molecular biologist and virologist at the
Institute of Immunology Centre de Recherche Public de la Santé/
Laboratoire National de Santé in Luxembourg. Her main research
interest is molecular epidemiology of infectious diseases,
especially influenza and other respiratory viruses.
References
1. Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team,
Dawood FS, Jain S, Finelli L, Shaw MW, Lindstrom S, et al. Emer-
gence of a novel swine-origin influenza A (H1N1) virus in humans.
N Engl J Med. 2009;360:2605­15. DOI: 10.1056/NEJMoa0903810
2. Hofshagen M, Gjerset B, Er C, Tarpai A, Brun E, Dannevig B, et al.
Pandemic influenza A(H1N1)v: human to pig transmission in Nor-
way? Euro Surveill. 2009;14:pii:19406.Medline
3. World Organisation for Animal Health. WAHID interface. Weekly
disease information [cited 2010 Mar 11]. http://www.oie.int/wahis/
public.php?page=weekly_report_index
4. Smith GJ, Vijaykrishna D, Bahl J, Lycett SJ, Worobey M, Pybus
OG, et al. Origins and evolutionary genomics of the 2009 swine-
origin H1N1 influenza A epidemic. Nature. 2009;459:1122­5. DOI:
10.1038/nature08182
5. Myers KP, Olsen CW, Gray GC. Cases of swine influenza in hu-
mans: a review of the literature. Clin Infect Dis. 2007;44:1084­8.
DOI: 10.1086/512813
6. Shinde V, Bridges CB, Uyeki TM, Shu B, Balish A, Xu X, et al.
Triple-reassortant swine influenza A (H1) in humans in the Unit-
ed States, 2005­2009. N Engl J Med. 2009;360:2616­25. DOI:
10.1056/NEJMoa0903812
7. Ayora-Talavera G, Cadavieco-Burgos JM, Canul-Armas AB. Se-
rologic evidence of human and swine influenza in Mayan persons.
Emerg Infect Dis. 2005;11:158­61.
8. Gray GC, McCarthy T, Capuano AW, Setterquist SF, Olsen CW,
Alavanja MC. Swine workers and swine influenza virus infections.
Emerg Infect Dis. 2007;13:1871­8.
9. Gregory V, Bennett M, Thomas Y, Kaiser L, Wunderli W, Matter
H, et al. Human infection by a swine influenza A (H1N1) virus in
Switzerland. Arch Virol. 2003;148:793­802. DOI: 10.1007/s00705-
002-0953-9
10. Newman AP, Reisdorf E, Beinemann J, Uyeki TM, Balish A, Shu
B, et al. Human case of swine influenza A (H1N1) triple reassortant
virus infection, Wisconsin. Emerg Infect Dis. 2008;14:1470­2. DOI:
10.3201/eid1409.080305
11. Ramirez A, Capuano AW, Wellman DA, Lesher KA, Setterquist SF,
Gray GC. Preventing zoonotic influenza virus infection. Emerg In-
fect Dis. 2006;12:996­1000.
12. Robinson JL, Lee BE, Patel J, Bastien N, Grimsrud K, Seal RF, et al.
Swine influenza (H3N2) infection in a child and possible community
transmission, Canada. Emerg Infect Dis. 2007;13:1865­70.
13. Brockwell-Staats C, Webster RG, Webby RJ. Diversity of influenza
viruses in swine and the emergence of a novel human pandemic in-
fluenza A (H1N1). Influenza Other Respi Viruses. 2009;3:207­13.
DOI: 10.1111/j.1750-2659.2009.00096.x
14. Pensaert M, Ottis K, Vandeputte J, Kaplan MM, Bachmann PA.
Evidence for the natural transmission of influenza A virus from wild
ducks to swine and its potential importance for man. Bull World
Health Organ. 1981;59:75­8.
15. Van Reeth K, Nicoll A. A human case of swine influenza virus infec-
tion in Europe­implications for human health and research. Euro
Surveill. 2009;14:pii:19124.
16. Ikonen N, Strengell M, Kinnunen L, Osterlund P, Pirhonen J, Bro-
man M, et al. High frequency of cross-reacting antibodies against
2009 pandemic influenza A(H1N1) virus among the elderly in Fin-
land. Euro Surveill. 2010;15:pii: 19478.
17. Miller E, Hoschler K, Hardelid P, Stanford E, Andrews N, Zambon
M. Incidence of 2009 pandemic influenza A H1N1 infection in Eng-
land: a cross-sectional serological study. Lancet. 2010;375:1100­8.
DOI: 10.1016/S0140-6736(09)62126-7
18. Centers for Disease Control and Prevention. Serum cross-reactive
antibody response to a novel influenza A (H1N1) virus after vacci-
nation with seasonal influenza vaccine; 2009. MMWR Morb Mortal
Wkly Rep. 2009;58:521­4.
19. Gatherer D. The 2009 H1N1 influenza outbreak in its historical con-
text. J Clin Virol. 2009;45:174­8. DOI: 10.1016/j.jcv.2009.06.004
20. World Health Organization. WHO manual on animal influenza diag-
nosis and surveillance [cited 2010 Jan 6]. http://www.wpro.who.int/
internet/resources.ashx/CSR/Publications/manual+on+animal+ai+
diagnosis+and+surveillance.pdf
21. Van Reeth K, Braeckmans D, Cox E, Van Borm S, van den Berg
T, Goddeeris B, et al. Prior infection with an H1N1 swine influ-
enza virus partially protects pigs against a low pathogenic H5N1
avian influenza virus. Vaccine. 2009;27:6330­9. DOI: 10.1016/j.
vaccine.2009.03.021
22. Kyriakis CS, Olsen CW, Carman S, Brown IH, Brookes SM, Van
Doorsselaere J, et al. Serologic cross-reactivity with pandemic
(H1N1) 2009 virus in pigs, Europe. Emerg Infect Dis. 2010:16:96­
9.
23. Capuano AW, Dawson JD, Gray GC. Maximizing power in seroepi-
demiological studies through the use of the proportional odds model.
Influenza Other Respi Viruses. 2007;1:87­93. DOI: 10.1111/j.1750-
2659.2007.00014.x
24. McCullagh P. Regression models for ordinal data. J R Statist Soc B.
1980;42:109­42.
25. European Centre for Disease Prevention and Control. European In-
fluenza Surveillance Network (EISN) [cited 2010 Mar 10]. http://
ecdc.europa.eu/en/activities/surveillance/EISN/Pages/home.aspx
410 Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 17, No. 3, March 2011
Swine Influenza Virus Antibodies in Humans
26. Laboratoire National de Santé. Sentinel surveillance of influenza
[cited 2010 Feb 20]. http://www.lns.public.lu/statistiques/grippe/
index.html
27. De Vleeschauwer AR, Van Poucke SG, Karasin AI, Olsen CW,
Van Reeth K. Original article: cross-protection between antigeni-
cally distinct H1N1 swine influenza viruses from Europe and North
America. Influenza and Other Respi Viruses. 2010. Epub ahead of
print [cited 2011 Jan 5]. http://onlinelibrary.wiley.com/doi/10.1111/
j.1750-2659.2010.00164.x/abstract
28. Neuzil KM. Pandemic influenza vaccine policy--considering the
early evidence. N Engl J Med. 2009;361:e59 [Epub 2009 Sep 10].
DOI: 10.1056/NEJMe0908224
29. Itoh Y, Shinya K, Kiso M, Watanabe T, Sakoda Y, Hatta M, et al. In
vitro and in vivo characterization of new swine-origin H1N1 influ-
enza viruses. Nature. 2009;460:1021­5.
30. Chen H, Wang Y, Liu W, Zhang J, Dong B, Fan X, et al. Serologic
survey of pandemic (H1N1) 2009 virus, Guangxi Province, China.
Emerg Infect Dis. 2009;15:1849­50.
31. Zhu FC, Wang H, Fang HH, Yang JG, Lin XJ, Liang XF, et al. A
novel influenza A (H1N1) vaccine in various age groups. N Engl J
Med. 2009;361:2414­23. DOI: 10.1056/NEJMoa0908535
32. Myers KP, Olsen CW, Setterquist SF, Capuano AW, Donham KJ,
Thacker EL, et al. Are swine workers in the United States at in-
creased risk of infection with zoonotic influenza virus? Clin Infect
Dis. 2006;42:14­20. DOI: 10.1086/498977
33. Olsen CW, Brammer L, Easterday BC, Arden N, Belay E, Baker I, et
al. Serologic evidence of H1 swine influenza virus infection in swine
farm residents and employees. Emerg Infect Dis. 2002;8:814­9.
Address for correspondence: Claude P. Muller, Institute of Immunology,
CRP-Santé/LNS, 20A, Rue Auguste Lumière, L-1950 Luxembourg;
email: claude.muller@lns.etat.lu
Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 17, No. 3, March 2011 411
