Analysis of cross reactivity of porcine sera in the haemagglutination inhibition assay using swine H1N1 or the new pandemic H1N1 influenza virus

G.E. Martín-Vallsa, M. Simon-Griféa, N. Busquetsa, I. Díaz,M. Martína, b, J. Casala,b, E. Mateua, b.

aCentre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Autònoma de Barcelona , Barcelona, Spain. bDepartament de Sanitat i d’Anatomia Animals, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Barcelona, Spain. *Email address:

Introduction

In April 2009, a new influenza A/H1N1 strain circulating in the human population was reported in Mexico. The new H1N1 virus was a triple swine/human/avian reassortant that afterwards spread pandemically. Fortunately, the virulence of the pandemic H1N1 (panH1N1) seems to be low but this episode made clear the need for a better surveillance of influenza A viruses infection. Swine influenza is most often caused by subtypes A/H1N1, A/H1N2 and A/H3N2. Hemagglutination inhibition test (HI) still is the serological assay of reference and it is assumed to be subtype-specific1. The main objective of the present work was to assess to what extent the use of different strains of a same subtype may affect the outcome of HI when examining swine serum samples.

Material and methods

Ninety-four serum samples were selected from a sera bank (2007-2009) corresponding to a cross-sectional study of swine influenza in Spain where sera had been tested in HI using anEuropean avian-like A/swine/Neth/Best/96 strain (NLH1N1)(GD laboratories, Deventer, Netherlands). The group of selected sera included negative and positive anti-NLH1N1 sera (1:320) that were re-tested in HI using the Spanish avian-like H1N1 A/swine/Spain /53207/2004 strain (ESH1N1) and the novel pandemic H1N1 (A/Catalonia/S2169/2009) strain (panH1N1) isolated from a human sample. Similarity of the predicted amino acid sequences of H1 were 89.3% between NLH1N1 and ESH1N1; 75.1% between NLH1N1 and panH1N1 and 71.5% between ESH1N1 and panH1N1. Sera were also examined using A/H1N2 and A/H3N2 (A/swine/Gent/7625/99 and A/swine/Neth/St.Oedenrode/96)(GD laboratories, Deventer, Netherlands) strains. Statistical analysis was done with Statdirect 2.7.5.

Results

Figure 1 show the comparative distribution of HI titres using the different H1N1 strains. Comparison of results of sera analyzed with NLH1N1 or ESH1N1 showed that both strains produced fairly similar titres (r=0.79; CI95%:0.70-0.85). In contrast, regression of log-transformed titres for NLH1N1 or ESH1N1 and panH1N1 was non significant, suggesting thatantibodies towards panH1N1 did not cross-react with other H1N1. No cross-reactivity between none of the A/H1N1 and either H1N2 or H3N2 was observed (data not shown). Interestingly, in 4 farms animals seropositive (1:40) against panH1N1 were found. In one of those farms (analysed in the first quarter of 2009) 8 animals were positive (Table 1); some of them reacted with other swine influenza strains.

Figure 1. Distribution HI titres against three different H1 influenza A strains: a) NLH1N1 vs. ESH1N1; b) ESH1N1 vs. panH1N1, c) panH1N1 vs NLH1N1.

Table 1. Results by HI with different A/influenza strains of the 8 pigs seropositive to panH1N1.

Pig / panH1 / NLH1 / ESH1 / H1N2 / H3N2
1 / 1:160 / 1:160 / 1:160 / 1:80 / Neg.
2 / 1:80 / 1:80 / 1:80 / Neg. / Neg.
3 / 1:80 / 1:80 / 1:80 / 1:80 / 1:160
4 / 1:80 / 1:80 / 1:160 / Neg. / Neg.
5 / 1:80 / 1:40 / 1:160 / Neg. / 1:80
6 / 1:80 / 1:80 / 1:40 / Neg. / 1:640
7 / 1:80 / 1:160 / 1:160 / 1:80 / 1:160
8 / 1:80 / 1:80 / 1:40 / Neg. / 1:320

Discussion

The present results indicate that panH1N1 has little or no cross-reactivity in terms of HI with other contemporary swine H1N1 viruses. However, the results also showed that, most probably, H1 viruses cross-reacting with the panH1N1 strain already existed before or at the same time of the emergence of that new pandemic strain. Considering the limited virulence of current panH1N1 it can not be discarded that similar or cross-reacting H1N1 virus circulated completely unnoticed in pig herds. Also, raise the question of how to interpret the HI results obtained with a given influenza strain.

References

1.Van Reeth, K. et al. Viral Inmunology.19;373-382 (2006).