Detection of quantitative trait loci for resistance/susceptibility to pseudorabies virus in swine

Gerald Reiner1, Elke Melchinger1, Marcela Kramarova1, Eberhardt Pfaff2, Matthias Büttner2, Armin Saalmüller2 and Hermann Geldermann1

Department of Animal Breeding and Biotechnology, University of Hohenheim, Garbenstraße 17, D-70593 Stuttgart, Germany1
Federal Research Centre for Virus Diseases of Animals, D-72076 Tübingen, Germany2

Author for correspondence: Gerald Reiner. Present address: Professur für Schweinekrankheiten, Justus-Liebig-Universität Giessen, Frankfurter Strasse 112, D-35392 Giessen, Germany. Fax +49 641 201854. e-mail gerald.reiner{at}vetmed.uni-giessen.de


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
This study describes genetic differences in resistance/susceptibility to pseudorabies virus (PrV) between European Large White and Chinese Meishan pigs, with a mapping of quantitative trait loci (QTL) obtained from a genome-wide scan in F2 animals. Eighty-nine F2 pigs were challenged intranasally at 12 weeks with 105 p.f.u. of the wild-type PrV strain NIA-3. For QTL analysis, 85 microsatellite markers, evenly spaced on the 18 porcine autosomes and on the pseudoautosomal region of the X chromosome, were genotyped. All pigs developed clinical signs, i.e. fever, from 3 to 7 days p.i. The pure-bred Large White pigs, the F1 and three-quarters of the F2 animals, but none of the Meishan pigs, developed neurological symptoms and died or were euthanized. QTLs for appearance/non-appearance of neurological symptoms were found on chromosomes 9, 5, 6 and 13. They explained 10·6–17·9% of F2 phenotypic variance. QTL effects for rectal temperature after PrV challenge were found on chromosomes 2, 4, 8, 10, 11 and 16. Effects on chromosomes 9, 10 and 11 were significant on a genome-wide level. The results present chromosomal regions that are associated with presence/absence of neurological symptoms as well as temperature course after intranasal challenge with NIA-3. The QTLs are in proximity to important candidate genes that are assumed to play crucial roles in host defence against PrV.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Pseudorabies virus (PrV), the causative agent of Aujeszky’s disease, is a neuroinvasive alphaherpesvirus with a wide host range, only excluding primates (Mettenleiter, 2000 ; Zuckermann, 2000 ). It involves the CNS, respiratory system and other major organs (Baskerville et al., 1973 ; Kluge et al., 1999 ). Aujeszky’s disease causes economic losses associated with reproduction failure and neonatal mortality in pigs. The severity of clinical symptoms is influenced by the age and immunological status of the animal, as well as the virulence of the virus and dose of exposure to the virus. Piglets from non-immune sows can suffer 100% mortality during the first 2 weeks. In older pigs, the disease is not lethal, but is characterized by severe depression, anorexia, pyrexia, ataxia, respiratory distress and abortion in sows (Baskerville, 1981 ; Kluge et al., 1999 ).

Indications of genetic differences in serum-neutralization titres of pigs after vaccination with pseudorabies vaccine and individual differences in cell-mediated and humoral immunity and in susceptibility to PrV in pigs were observed by Rothschild et al. (1984) , Meeker et al. (1987a , b ) and Hessing et al. (1994 , 1995 ). However, no quantitative trait loci (QTL) have been identified to date for resistance against PrV. The objectives of this study were to map QTLs in a genome-wide scan for resistance/susceptibility to PrV in informative F2 pig families and to indicate candidate genes that are probably involved in resistance to PrV in swine.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
Three Large White boars and two Meishan sows were used as founders for four F1 boars and seven F1 sows. From nine litters, 89 F2 individuals were generated. Blood samples were taken for isolation of genomic DNA. Litters were housed and fed under standardized conditions at the experimental station ‘Unterer Lindenhof’ of the University of Hohenheim. At 12±2 weeks, the F2 animals were transported to the Federal Research Centre for Virus Diseases of Animals (BFAV) in Tübingen. Additionally, eight pure-bred Large White, five pure-bred Meishan and nine F1 pigs of the same age were included. After 1 week of acclimatization, they were challenged intranasally with 105 p.f.u. of the wild-type PrV strain NIA-3 (McFerran & Dow, 1975 ). Rectal temperatures were measured and animals were screened daily for the onset of neurological symptoms, such as trembling, incoordination, ataxia, paralysis, circling and paddling. Pigs that developed neurological signs were euthanized under a barbiturate anaesthesia.

Eighty-five microsatellite markers were selected from the map produced by Rohrer et al. (1996) (http://sol.marc.usda.gov/) based on their position, ease of scoring and informativity. Markers were spaced evenly on the 18 porcine autosomes and on the pseudoautosomal region of the X chromosome (SSCX) (Fig. 2). The maximum interval based on the USDA map was 40 cM. Linkage was analysed with the software package CRIMAP (Green et al., 1990 ) according to the guidelines of Keats et al. (1991) . Sex-averaged maps were constructed.

QTL analysis was done according to an interval mapping strategy (Haley et al., 1994 ) with a monolocus regression analysis. The statistical model included effects of sex, age at challenge and family. Chromosome-specific empirical threshold values of the F statistic were estimated via permutation test (Churchill & Doerge, 1994 ). The 5% genome-wide, 10% genome-wide and 5% chromosome-wide thresholds were estimated as 8·8, 7·9 and 5·6.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Phenotypic differences between challenged pigs
All challenged pigs developed clinical signs, i.e. fever, from 3 to 7 days p.i. The pure-bred Large White pigs had neurological symptoms at days 5 and 6 and were euthanized. The F1 and three-quarters of the F2 pigs developed neurological signs 1 day later. All Meishan pigs and a quarter of the F2 pigs did not develop any neurological symptoms and recovered until 8 to 9 days p.i. The rise in temperature in F2 animals started 2 days p.i. (Fig. 1). At day 3, two groups with different fever courses could be distinguished, one showing a quick rise, reaching temperatures of about 40·8 °C, and a second group of individuals that stayed below 40·5 °C until day 6. As shown in Table 1, about three-quarters of the F2 individuals belonged to the high-temperature group. Temperature profiles and the appearance of neurological symptoms were not correlated. Results of clinical traits in F2 animals after intranasal challenge are given in Table 2.



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Fig. 1. Profiles of rectal temperatures of F2 pigs after intranasal challenge with 105 p.f.u. of the PrV strain NIA-3 at the age of 12 weeks. Asterisks indicate that differences in temperature were statistically significant (P<0. 001). Groups: A ({bullet}), small increase in temperature, no neurological symptoms (5·6% of the F2 pigs); B ({circ}), large increase in temperature, no neurological symptoms (23·6%); C ({blacksquare}), small increase in temperature, neurological symptoms (20·2%); D ({square}), large increase in temperature, neurological symptoms (50·6%).

 

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Table 1. Presence of neurological symptoms and temperature types in F2 pigs

 

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Table 2. Clinical traits in F2 animals after intranasal challenge with PrV

 
QTL effects
QTL effects on rectal temperatures after challenge with PrV were located on chromosomes 2, 4, 8, 11 and 16 (Table 3; Fig. 2). QTLs affecting the appearance/non-appearance of neurological symptoms and the day of onset of neurological symptoms were found on chromosomes 5, 6, 9 and 13 (Table 3; Fig. 2). F-ratio curves for the QTLs are shown in Fig. 3. Additive as well as dominant QTL effects were calculated (Table 3). Meishan alleles on SSC9 and SSC6 were associated with the absence of neurological symptoms. However, regarding the effects on SSC5 and SSC13, Large White alleles corresponded to smaller neurological signs. Multivariate analysis of the QTL effects on SSC9, SSC6 and SSC5 on appearance/non-appearance of neurological symptoms explained 35% of F2 phenotypic variance, with a multiple correlation of 0·59 (P<=0·0001; Table 4).


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Table 3. QTLs with effects on clinical signs after intranasal challenge of F2 pigs

 


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Fig. 2. Genome-wide mapping results for QTLs on body temperature and neurological symptoms in F2 pigs after intranasal challenge at the age of 12 weeks with 105 p.f.u. of the PrV strain NIA-3. QTLs are indicated on the chromosomes. Statistical thresholds for chromosome-wide and genome-wide significance are indicated. The scan included all autosomes and the pseudoautosomal region of the X chromosome, including 85 microsatellite markers, genotyped in 89 F2 individuals.

 


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Fig. 3. F-ratio curves of QTLs for appearance/non-appearance of neurological symptoms after intranasal challenge with 105 p.f.u. of the PrV strain NIA-3 on SSC9, SSC6 and SSC5. The x-axis indicates the relative positions of microsatellite markers on the chromosome in Kosambi cM. The y-axis represents the F ratio. Horizontal dotted lines indicate threshold values for statistical significance on the P<0·05 genome-wide (***), P<0·10 genome-wide (**) and P<0·05 chromosome-wide (*) levels.

 

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Table 4. Multiple analysis of the effects of QTLs on SSC9, SSC6 and SSC5 on appearance/non-appearance of neurological signs

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
A challenge test with 105 p.f.u. of the highly virulent NIA-3 strain revealed a significant difference in susceptibility/resistance of Meishan and Large White pigs against PrV. All pigs developed fever from day 2/3 p.i. to day 8/9 p.i. Neurological signs were visible in 100% of the Large White pigs tested, but did not appear in Meishan pigs. A ratio of susceptible:resistant animals of 3:1 was observed in F2 offspring of these founder breeds, indicating dominant inheritance. These differences between founder breeds motivated a QTL-mapping experiment in an F2 generation to search for effects on appearance/non-appearance of neurological symptoms after infection with PrV. Earlier studies had reported genetic differences in serum-neutralization titres of individual pigs after vaccination with pseudorabies vaccine (Rothschild et al., 1984 ) and individual differences in cell-mediated and humoral immunity and in susceptibility to PrV (Meeker et al., 1987a , b ; Hessing et al., 1994 , 1995 ), but there was no information on QTL mapping of such differences. The two novel sets of genome-wide significant QTLs found in this study point to gene effects on (i) the appearance/non-appearance of neurological symptoms and (ii) QTLs for temperature course after challenge with PrV. The neurological signs are associated mainly with QTLs located on SSC9, SSC6 and SSC5. QTLs with effects on the temperature profile were mapped on chromosomes 2, 4, 8, 11 and 16 and some other chromosomes. They seem to be independent of QTLs for neurological symptoms.

Due to the limited number of F2 animals, QTL positions within chromosomes could not be mapped very precisely. Thus, the identification of candidate genes can only be assumed very roughly. Major QTLs on SSC9 and SSC6 are linked with the loci PRR1 [polio-related receptor 1, HveC (herpes virus entry protein C); Geraghty et al., 1998 ] and PRR2 (HveB; Eberle et al., 1995 ). Both receptor proteins are involved in adsorption and penetration of PrV to the cell in rodent models. Initiation of infection by alphaherpesviruses requires a cascade of interactions between different virus and cellular membrane components (Karger & Mettenleiter, 1996 ). Interaction of the receptors with virus glycoprotein gE seems to influence markedly the neurological spread of the infection (Kimman et al., 1992 ; Kritas et al., 1994 ; Husak et al., 2000 ). To date, the effects of PRR1 and PRR2 on porcine PrV infection are unknown, and the linked QTLs presented in our study indicate the need for more specific investigation of these genes. Furthermore, the absence of QTLs in the region of the MHC must be mentioned, since Favoreel et al. (1999) assumed an important role of this gene complex in resistance/susceptibility to PrV. However, a number of further components of the immune system can influence resistance/susceptibility to alphaherpesviruses (Sin et al., 1999 ). Specific immunology against herpesviruses seems to be sustained by the IL-12/IFN-{gamma} pathway (Grob et al., 1999 ; Zuckermann, 2000 ). The IL-12 gene is located within an interleukin cluster on SSC2, close to the marker Swr349, a region associated with a QTL on temperature course. Further QTLs are linked to the IFN-{gamma} locus (SSC5) and the locus for an interferon receptor (SSC13). Thus, more specific research should include the candidate genes (PRR1, PRR2, IFN-{gamma}, IL-12, interferon receptor) and analyse their role in porcine PrV. Since our study elucidates genetic differences in resistance/susceptibility to PrV between Meishan and Large White pigs, these genetically diverse breeds are informative in elucidation of the role of host defence against PrV in swine.


   Acknowledgments
 
Primers for analysis of microsatellites were kindly provided by Dr G. A. Rohrer, USDA, USA. The Meishan pigs were obtained from Eubrid, Boxmeer, The Netherlands.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 9 August 2001; accepted 18 September 2001.



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