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The Veterinary Journal 172 (2006) 377–381

TheVeterinary Journal

Short communication

Evidence of the concurrent circulation of H1N2, H1N1 andH3N2 influenza A viruses in densely populated pig areas in Spain

Jaime Maldonado a,*, Kristien Van Reeth b, Pere Riera a, Marta Sitja a,Narcıs Saubi a, Enric Espuna a, Carlos Artigas a

a Laboratorios HIPRA, S. A., Av. La Selva No. 135, Amer, 17170 Girona, Spainb Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

Abstract

This paper reports on a serological and virological survey for swine influenza virus (SIV) in densely populated pig areas in Spain.

The survey was undertaken to examine whether the H1N2 SIV subtype circulates in pigs in these areas, as in other European

regions. Six hundred sow sera from 100 unvaccinated breeding herds across Northern and Eastern Spain were examined using

haemagglutination inhibition (HI) tests against H1N1, H3N2 and H1N2 SIV subtypes. Additionally, 225 lung samples from pigs

with respiratory problems were examined for the presence of SIV by virus isolation in embryonated chicken eggs and by a commer-

cial membrane immunoassay. The virus isolates were further identified by HI and RT-PCR followed by partial cDNA sequencing.

The HI test on sera revealed the presence of antibodies against at least one of the SIV subtypes in 83% of the herds and in 76.3% of

the animals studied. Of the 600 sow sera tested, 109 (18.2%), 60 (10%) and 41 (6.8%) had SIV antibodies to subtype H1N2 alone,

H3N2 alone and H1N1 alone, respectively. Twelve H3N2 viruses, 9 H1N1 viruses and 1 H1N2 virus were isolated from the lungs of

pigs with respiratory problems. The analysis of a 436 nucleotide sequence of the neuraminidase gene from the H1N2 strain isolated

further confirmed its identity. Demonstrably, swine influenza is still endemic in the studied swine population and a new subtype, the

H1N2, may be becoming established and involved in clinical outbreaks of the disease in Spain.

� 2005 Elsevier Ltd. All rights reserved.

Keywords: Influenza A viruses; H1N2; Swine; Influenza; Flu; Spain

Surveillance of swine influenza (SI) viruses is impor-

tant because new influenza viruses and influenza virus

reassortants regularly emerge in pig populations, and be-

cause there are considerable antigenic and genetic differ-

ences between swine influenza viruses (SIVs) in different

geographic regions. Monitoring and characterization ofSIV in pig producing regions is needed for the adequate

control and diagnosis of infection. Furthermore, there

are concerns that pigs may serve as intermediate hosts

for the introduction of new influenza viruses into the hu-

man population (Brown et al., 1998).

1090-0233/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.tvjl.2005.04.014

* Corresponding author. Tel.: +34 972 43 06 55; fax: +34 972 43 06

61.

E-mail address: jmg@hipra.com (J. Maldonado).

The H1N1 and H3N2 SIV subtypes have been enzo-

otic in several swine producing countries in Europe for

more than 20 years (Brown, 2000). A third subtype,

H1N2, was first isolated in Great Britain in 1994 (Brown

et al., 1995). Thereafter, the H1N2 virus subtype has

also been found in France, Belgium, Italy and Germany,where it appears to co-circulate with H1N1 and H3N2

viruses (Gourreau et al., 1994; Van Reeth et al., 2000;

Marozin et al., 2002; Schrader and Suss, 2003).

In Spain, SIV subtypes H1N1 and H3N2 have been

circulating since the mid 1980s. Both H1N1 (Plana

Duran et al., 1984) and H3N2 (Castro et al., 1988)

SIV strains have been isolated from pigs with acute

respiratory problems. Serological examinations offattening pigs between 1987 and 1989 revealed high

378 J. Maldonado et al. / The Veterinary Journal 172 (2006) 377–381

seroprevalences for both H1N1 (between 72.8% and

78.5% seropositive pigs) and H3N2 (between 61.6%

and 62.5% seropositive pigs) (Yus et al., 1989, 1992).

Recent reports point towards a continuous circulation

during the 1990s of H1N1 and H3N2 SIV in the Span-

ish pig population (Oliveira et al., 1999; Gutierrez-Mar-tin et al., 2000). However, there is little information on

the current seroprevalences of H1N1 and H3N2 sub-

type influenza viruses. Moreover, it is unknown

whether H1N2 SIV viruses are circulating in Spain, as

neither virus isolation nor prevalence of SIV subtype

specific antibodies have been recently reported.

In this paper, we report on a recent virological and

serological survey for different SIV subtypes in a repre-sentative part of the Spanish swine population. It was

the purpose of this survey to determine the prevalence

of H1N2 virus antibodies in the studied population,

and to compare it with that of H1N1 and H3N2 anti-

bodies. We also took the opportunity to subtype SIV

isolates from acute respiratory disease outbreaks.

Farm selection followed the criteria established by

the European Surveillance Network for Influenza in Pigs(ESNIP group). Breeding herds were included in the

study when they met the inclusion criteria of being lo-

cated in the most densely populated pig areas in Spain

and not having had vaccination programme against

SIV (confirmed by telephone interviews with the farm-

ers). Vaccination against SIV in Spain is neither compul-

sory nor common practice.

The number of samples was calculated from an esti-mated population of 875,000 females, with 4% precision

at a 95% confidence level (Epi Info software package,

version 5.01). Serological testing was performed on

600 sow sera from 100 conventional two-site operations

(six samples per unit including sows of all parities) lo-

cated in the four major Spanish swine producing auton-

omous regions (Aragon, Castilla and Leon, Catalonia

and Murcia) and distributed around 22 provinces inNorthern and Eastern Spain (accounting for 65% of

the national pig inventory). Sera were collected during

the period February–June 2003. On arrival at the labo-

ratory, sera were separated by centrifugation, split and

stored in vials at �20 �C until analysed.

Haemagglutination inhibition (HI) tests against H1N1,

H3N2 and H1N2 subtypes were performed using stan-

dard methods (Webster and Krauss, 2002). The SIVstrains Sw/Spain/45304/2003(H1N1), Sw/Spain/46356/

2003(H3N2) and Sw/Scotland/410440/94(H1N2) were

used as antigens. Two-fold serum dilutions were tested

starting at a dilution of 1/20. The results were recorded

as the log2 of the HI titre, and the geometric mean titre

(GMT) for each virus subtype was calculated. A farm

was classified as positive and assigned to a SIV subtype

when at least one of the six sows had HI titres above thecut-off value against 1 of the SIV subtypes alone, regardless

of the presence of individuals showing mixed infections.

In the virological surveillance, diagnostic specimens

were examined. Between November 2001 and April

2004, 225 pig lungs from unrelated growing-finishing

pig units (housing an average of 800 pigs per unit) were

submitted to the Veterinary Diagnostic Centre, Labora-

torios HIPRA, S.A. (Girona, Spain) for the aetiologicaldiagnosis of pneumonia. The herds of origin (located all

around Spain) were experiencing acute episodes of respi-

ratory disease, mainly at the end of the finishing period.

Affected animals showed clinical signs and macroscopic

lesions suggestive of acute viral pneumonia. Bacteriolog-

ical and virological analyses were performed to detect

the most relevant respiratory pathogens for swine (data

not shown). Samples (lung and bronchial tissues) forSIV investigation were systematically collected.

Ten per cent lung tissue homogenates were examined

for the presence of SIV antigen by a commercial mem-

brane enzyme immunoassay (EI) (BD Directigen Flu

A; Becton Dickinson Microbiology Systems) and for

virus isolation by inoculation in 9- to 11-day old spe-

cific-pathogen-free embryonated chicken eggs (ECE),

as described by Swenson et al. (2001).Allantoic fluids were harvested after 3–4 days of incu-

bation at 37 �C. The presence of SIV was determined in

a haemagglutination (HA) assay with 0.5% chicken red

blood cells. To determine the subtype of SIV isolates,

allantoic fluids were examined in a HI assay with swine

hyperimmune sera using standard methods (Webster

and Krauss, 2002) and by single-step reverse transcrip-

tion-polymerase chain reaction (RT-PCR). RNA wasextracted from 200 lL of allantoic fluids, by using the

RNeasy Protect Mini Kit (QIAGEN).

PCR amplification conditions and primers designed

to amplify partial sequences of the H1, H3, N1 and

N2 SIV genes were adapted from Chiapponi et al.

(2003). Briefly, 0.6 lM of each primer and 5 lL of the

extracted RNA were added to four separate RT-PCR

mixtures (one for each target) (OneStep RT-PCR Kit,QIAGEN) to obtain final volumes of 50 lL. The follow-

ing conditions were set up in a Px2 Thermal Cycler

(Thermo Electronic Corporation): the first strand

cDNA synthesis was conducted for 30 min at 50 �C.

After denaturation for 15 min at 95 �C, the samples were

submitted to 40 cycles of PCR amplification as follows:

1 min at 95 �C, 1 min at 50 �C and 1 min at 72 �C. The

final extension step was for 7 min at 72 �C. In all PCRreactions, predefined SIV-positive, -negative and non-

template controls were tested simultaneously with the

field isolates. PCR products were analysed by agarose

gel electrophoresis.

To further confirm the identity of some of the SIV

isolates, selected PCR products (N2 fragments) were se-

quenced using an ABI 373 DNA sequencer, together

with the Taq dye-Deoxy terminator cycle sequencingkit (Applied Biosystems). Comparative nucleotide and

amino acid sequence analysis were conducted using

J. Maldonado et al. / The Veterinary Journal 172 (2006) 377–381 379

software tools from the National Centre for Biotechnol-

ogy Information (NCBI, Maryland, USA [Available

from: http://www.ncbi.nlm.nih.gov/]).

The HI test revealed the presence of antibodies

against at least one of the SIV subtypes in 83% of the

herds and 76.3% of the animals studied. Of the 600sow sera tested, 109 (18.2%), 60 (10%) and 41 (6.8%)

had SIV antibody to subtype H1N2 alone, H3N2 alone

and H1N1 alone, respectively. Mixed infections were de-

tected in 41.3% of the animals tested. Most were housed

in farms in which the identification of the dominant sub-

type of circulating virus was not possible. Table 1 sum-

marizes the HI results at the sow and farm levels.

Twenty-two SIV lung samples (9.8%) were found tobe positive for SIV by isolation. All tested positive in

the EI, HI and PCR tests, and total agreement between

HI and PCR allowed the classification of the isolates as

being of the H3N2 (12 isolates), H1N1 (9 isolates) and

H1N2 (1 isolate) subtypes.

The H1N2 virus strain A/sw/Polenino/40564/

02(H1N2) was isolated from the lungs of a 5-month-old

fattening pig, during a typical outbreak of acute respira-tory disease. No other relevant pathogen from that partic-

ular sample was detected or isolated. The affected farm

had 1600 pigs and morbidity and mortality rates during

the respiratory disease outbreak were 50% and 1%,

respectively. Alignment of the N2 gene partial sequences

obtained from isolate A/sw/Polenino/40564/02(H1N2)

with those of the most closely related Italian and French

H1N2 strains available in electronic data bases (Gen-Bank, EMBL, DDBJ and PDB) (Accession Nos.:

AJ412696, AJ412705, AJ412701, AJ412693, AJ412698,

AJ412695, AJ412694 and AJ412704) revealed 95–97%

homology. Deduced protein sequences from both the Ital-

ian A/swine/Italy/1081/00(H1N2) and the French A/

swine/Cotes d�Armor/790/97(H1N2) strains (first and sec-

ond places in the nucleotide–nucleotide matching list of

the BLAST analysis) showed 97% homology with the A/sw/Polenino/40564/02(H1N2) isolate (Fig. 1).

Table 1

Numbers of individual animals (n = 600) and herds (n = 100) with HI antibo

(Spain, 2003)

Virus subtype(s) Number and percenta

Individual sows

None (negative to all 3 subtypes) 142b

H1N1 only 41

H3N2 only 60

H1N2 only 109

H1N1 + H3N2 40

H3N2 + H1N2 58

H1N1 + H1N2 80

H1N1 + H3N2 + H1N2 70

a The cut-off value for HI was 1/40.b Cumulative number of negative sows housed in positive and negative he

Our data show that SIVs of H1N1 and H3N2 subtype

have remained endemic in the Spanish swine population.

A new finding is that the recently emerged H1N2 SIV is

widespread in Spain and that it is involved in respiratory

outbreaks, as is the case in other European countries

(Van Reeth et al., 2000; Marozin et al., 2002; Schraderand Suss, 2003). Our serology results may raise the ques-

tion of whether the positive reactions to the H1N2 virus

in the HI test may in part be due to serological cross-

reaction with the H1N1 subtype or vice versa. However,

in experimental infection studies with both a single sub-

type and a combination of two SIV subtypes it has been

clearly demonstrated that there is no serological cross-

reaction between H1N1, H1N2 and H3N2 subtypes inthe HI test (Van Reeth et al., 2003). It is likely therefore

that those animals in this study with HI antibodies

against H1N1 or H1N2 have been previously infected

with the respective subtypes.

In our study, the serological determination of the

dominant serotype infecting a high proportion of the

tested sera was not feasible. It has recently been demon-

strated that when field serum contains significantamounts of anti-SIV antibody to more than one subtype

after natural infections, it cannot be classified in a reli-

able way by using current HI methods (Long et al.,

2004). This could be the situation in this study, as the

seroprevalence rates for H1N1, H3N2 and H1N2 SIV

observed suggest that the three subtypes are circulating

concurrently and cause mixed infections.

SIV was isolated from 9.8% of the 225 lung samplestested. This cannot be considered as a prevalence rate

as it is the result of a diagnostic exercise rather than a

prevalence survey. In other surveys of acute respiratory

disease outbreaks in the Netherlands, SIVs were isolated

from 45% of the acute respiratory disease outbreaks

examined (Loeffen et al., 1999) and similar SIV isolation

rates have been reported in Belgium (K. Van Reeth, per-

sonal communication) and in the US (Choi et al., 2003).It must be mentioned, therefore, that some of the

dies to one single SIV subtype or to combinations of 2 or 3 subtypes

ge of positivesa

(%) Herds (%)

23.7 17 17.0

6.8 9 9.0

10.0 20 20.0

18.2 21 21.0

6.6 6 6.0

9.7 6 6.0

13.3 5 5.0

11.7 16 16.0

rds.

(a)

(b)

Fig. 1. Partial nucleotide and deduced amino acid sequences for the neuraminidase gene of the virus strain A/sw/Polenino/40564/02(H1N2). The

nucleotide sequence (436 nucleotides) is shown in the top lines (a). Alignment with the predicted amino acid sequences for A/swine/Italy/1081/

00(H1N2) (Accession No. AJ412696) and A/swine/Cotes d�Armor/790/97(H1N2) (Accession No. AJ412705) was performed by ClustalW programme

(http://www.ebi.ac.uk/clustalw/): Shaded areas denote sequence differences.

380 J. Maldonado et al. / The Veterinary Journal 172 (2006) 377–381

diagnostic submissions in the present study were accom-

panied by rather vague descriptions of the clinical symp-

toms, and only the known acute cases of respiratory

disease were examined in the studies mentioned above.Other factors that may account for the rather low SIV

isolation rates in the present study are suboptimal con-

servation and autolysis of part of the lung tissue sam-

ples. Although direct PCR could give a better

prevalence estimation of SIV infection on autolytic sam-

ples, this was not the objective of our survey.

With the limited clinical information available, we

cannot assume that the SIVs isolated played a centralrole in the course of the studied outbreaks. Furthermore,

other bacteria and viruses were also present in some of

the SIV-positive lungs (data not shown). It should be

mentioned nevertheless, that SIV is recognized as an

important contributor in the aetiology of the porcine

respiratory disease complex, infecting alone or in combi-

nation with other pathogens (Thacker et al., 2001).

The origin of the strain A/sw/Polenino/40564/02(H1N2) remains elusive, but genetic analyses of the

N2 suggest that this strain is related to H1N2 viruses

isolated in France and in Italy since the late 1990s.

Based on the antigenic and genetic relationships of

viruses isolated in France, Italy and the UK, Marozin

et al. (2002) stated that H1N2 viruses were introduced

into continental Europe from the UK. However,

sequencing of the internal genes is required to furtherdetermine the origin of the Spanish H1N2 strain, as re-

cent studies have demonstrated extensive antigenic and

genetic heterogeneity among H1N2 viruses (Marozin

et al., 2002).

Results obtained in the present study highlight the

need for continuous surveillance for SIVs in pig produc-

ing areas, as new and potentially pathogenic SIV sub-

types readily emerge. Also, this study will contributesignificantly to the task of the ESNIP group in monitor-

ing the evolution of SI in Europe during the next years.

All the data collected and viruses isolated until now, and

the obtained ones in a future, will be deposited in the

virus bank and the electronic database for further stud-

ies on molecular epidemiology, immunology and genet-

ics of SI in Europe (http://www.esnip.wur.nl/).

Acknowledgements

The authors thank Romney Jackson for helpful lan-

guage revision. Technical assistance of Corona Vinals

is gratefully acknowledged. This research was supported

by Laboratorios HIPRA S. A., Amer, Gerona, Spain

and ESNIP (EC concerted action, 6th Framework Pro-gramme, QLK2-CT-2000-01636).

References

Brown, I.H., Chakraverty, P., Harris, P.A., Alexander, D.J., 1995.

Disease outbreaks in pigs in Great Britain due to an Influenza A

virus of H1N2 subtype. The Veterinary Record 136, 328–329.

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. Journal of General Virology 79, 2947–

2955.

J. Maldonado et al. / The Veterinary Journal 172 (2006) 377–381 381

Brown, I.H., 2000. The epidemiology and evolution of Influenza

viruses in pigs. Veterinary Microbiology 74, 29–46.

Castro, J.M., del Pozo, M., Simarro, I., 1988. Identification of H3N2

Influenza virus isolated from pigs with respiratory problems in

Spain. The Veterinary Record 122, 418–419.

Chiapponi, C., Fallacara, F., Foni, E., 2003. Subtyping of H1N1,

H1N2 and H3N2 swine Influenza viruses by two multiplex RT-

PCR. In: Proceedings of the 4th International Symposium on

Emerging and Re-emerging Pig Diseases, Rome, Italy, p. 257.

Choi, Y.K., Goyal, S.M., Joo, H.S., 2003. Retrospective analysis of

etiologic agents associated with respiratory diseases in pigs.

Canadian Veterinary Journal 44, 735–737.

Gourreau, J.M., Kaiser, C., Valette, M., Douglas, A.R., Labie, J.,

Aymard, M., 1994. Isolation of two H1N2 Influenza viruses from

swine in France. Archives of Virology 35, 365–382.

Gutierrez-Martin, C.B., Rodriguez-Delgado, O., Alvarez-Nistal, D.,

De La Puente-Redondo, V., Garcia-Rioja, A.F., Martin-Vicente,

J., Rodriguez Ferri, E.F., 2000. Simultaneous serological evidence

of Actinobacillus pleuropneumoniae, PRRS, Aujeszky�s disease and

Influenza viruses in Spanish finishing pigs. Research in Veterinary

Science 68, 9–13.

Loeffen, W.L., Kamp, E.M., Stockhofe-Zurwieden, N., van Nieuws-

tadt, A.P., Bongers, J.H., Hunneman, W.A., Elbers, A.R., Baars,

J., Nell, T., van Zijderveld, F.G., 1999. Survey of infectious agents

involved in acute respiratory disease in finishing pigs. The

Veterinary Record 31, 123–129.

Long, B.C., Goldberg, T.L., Swenson, S.L., Erickson, G., Scherba, G.,

2004. Adaptation and limitations of established hemagglutination

inhibition assays for the detection of porcine anti-swine influenza

virus H1N2 antibodies. Journal of Veterinary Diagnostic Investi-

gation 16, 264–270.

Marozin, S., Gregory, V., Cameron, K., Bennett, M., Valette, M.,

Aymard, M., Foni, E., Barigazzi, G., Lin, Y., Hay, A., 2002.

Antigenic and genetic diversity among swine Influenza A H1N1

and H1N2 viruses in Europe. Journal of General Virology 83, 735–

745.

Oliveira, J., Guitian, J., Yus, E., Sanjuan, M.L., 1999. Description of

serological results against several strains of Influenza virus in a

population of slaughter pigs in NW Spain. In: Proceedings of the

Symposium on Animal Influenza Viruses, Gent, Belgium, pp. 38–

39.

Plana Duran, J., Vayreda, M., Vila, X., Marrull, L., 1984. Isolation for

the first time in Spain of the swine Influenza virus. In: Proceedings

of the 8th International Pig Veterinary Society Congress, Gent,

Belgium, p. 62.

Schrader, C., Suss, J., 2003. Genetic characterization of a porcine

H1N2 Influenza virus strain isolated in Germany. Intervirology 46,

66–70.

Swenson, S.L., Vincent, L.L., Lute, B.M., Janke, B.H., Lechtenberg,

K.E., Landgraf, J.G., Schmitt, B.J., Kinker, D.R., McMillen, J.K.,

2001. A comparison of diagnostic assays for the detection of type A

swine Influenza virus from nasal swabs and lungs. Journal of

Veterinary Diagnostic Investigation 13, 36–42.

Thacker, E.L., Thacker, B.J., Janke, B.H., 2001. Interaction between

Mycoplasma hyopneumoniae and swine influenza virus. Journal of

Clinical Microbiology 39, 2525–2530.

Van Reeth, K., Brown, I.H., Pensaert, M., 2000. Isolations of H1N2

Influenza A virus from pigs in Belgium. The Veterinary Record

146, 588–589.

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.

Webster, R.G., Krauss, S. (Eds.), 2002. World Health Organization

Manual on Animal Influenza Diagnosis and Surveillance. World

Health Organization, Geneva, Switzerland.

Yus, E., Laviada, M.D., Moreno, L., Castro, J.M., Escribano, J.M.,

Simarro, I., 1989. The prevalence of antibodies to Influenza virus

and respiratory corona virus in fattening pigs in Spain. Zentralblatt

fur Veterinarmedizin Reihe B 36, 551–556.

Yus, E., Sanjuan, M.L., Garcia, F., Castro, J.M., Simarro, I., 1992.

Influenza A viruses: epidemiologic study in fatteners in Spain

(1987–89). Zentralblatt fur Veterinarmedizin Reihe B 39, 113–118.