Detection of quantitative trait loci for resistance

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did not develop any neurological symptoms and recovered until 8 to 9 days ..... Percentage of F. # variance. Single analysis. Multiple analysis. QTL r. %s F2. P.

Journal of General Virology (2002), 83, 167–172. Printed in Great Britain ...................................................................................................................................................................................................................................................................................

Detection of quantitative trait loci for resistance/susceptibility to pseudorabies virus in swine Gerald Reiner,1 Elke Melchinger,1 Marcela Kramarova,1 Eberhardt Pfaff,2 Matthias Bu$ ttner,2 Armin Saalmu$ ller2 and Hermann Geldermann1 1 2

Department of Animal Breeding and Biotechnology, University of Hohenheim, Garbenstraße 17, D-70593 Stuttgart, Germany Federal Research Centre for Virus Diseases of Animals, D-72076 Tu$ bingen, Germany

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 10n6–17n9 % 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 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 nonimmune 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, Author for correspondence : Gerald Reiner. Present address : Professur fu$ r Schweinekrankheiten, Justus-Liebig-Universita$ t Giessen, Frankfurter Strasse 112, D-35392 Giessen, Germany. Fax j49 641 201854. e-mail gerald.reiner!vetmed.uni-giessen.de

0001-8031 # 2002 SGM

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 F pig # families and to indicate candidate genes that are probably involved in resistance to PrV in swine.

Methods Three Large White boars and two Meishan sows were used as founders for four F boars and seven F sows. From nine litters, 89 F " " # individuals were generated. Blood samples were taken for isolation of genomic DNA. Litters were housed and fed under standardized BGH

G. Reiner and others conditions at the experimental station ‘ Unterer Lindenhof ’ of the University of Hohenheim. At 12p2 weeks, the F animals were # transported to the Federal Research Centre for Virus Diseases of Animals (BFAV) in Tu$ bingen. Additionally, eight pure-bred Large White, five pure-bred Meishan and nine F pigs of the same age were included. After " 1 week of acclimatization, they were challenged intranasally with 10& 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  (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. Chromosomespecific 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 8n8, 7n9 and 5n6.

Results 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 F and three-quarters of the F pigs developed neurological " # signs 1 day later. All Meishan pigs and a quarter of the F pigs # did not develop any neurological symptoms and recovered until 8 to 9 days p.i. The rise in temperature in F 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 40n8 mC, and a second group of individuals that stayed below 40n5 mC until day 6. As shown in Table 1, about three-quarters of the F individuals belonged to # the high-temperature group. Temperature profiles and the appearance of neurological symptoms were not correlated. Results of clinical traits in F animals after intranasal challenge # are given in Table 2. 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, reBGI

*

*

*

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 ($), small increase in temperature, no neurological symptoms (5n6 % of the F2 pigs) ; B (#), large increase in temperature, no neurological symptoms (23n6 %) ; C ( ), small increase in temperature, neurological symptoms (20n2 %) ; D ( ), large increase in temperature, neurological symptoms (50n6 %).

Table 1. Presence of neurological symptoms and temperature types in F2 pigs Eighty-nine F individuals were challenged intranasally with 10& p.f.u. # of the PrV strain NIA-3 at the age of 12 weeks. Percentages are given in parentheses. The ‘ low ’ temperature type showed a mean rectal temperature on days 2–5 p.i. below 40n5 mC. The ‘ high ’ temperature type showed a mean rectal temperature on days 2–5 p.i. above 40n8 mC. Temperature type Neurological symptoms No Yes … Totals

Low

High

Totals

5 (19n2) 18 (28n6) 23 (25n8)

21 (80n8) 45 (71n4) 66 (74n2)

26 (29n2) 63 (70n8) 89 (100)

garding 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 F phenotypic variance, with a multiple # correlation of 0n59 (P  0n0001 ; Table 4).

Discussion A challenge test with 10& p.f.u. of the highly virulent NIA3 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 F #

QTLs for PrV resistance/susceptibility

Table 2. Clinical traits in F2 animals after intranasal challenge with PrV Animals were challenged with 10& p.f.u. of PrV strain NIA-3 at the age of 12 weeks.

Trait Temperature (mC) : Day 0 p.i.* Day 1 p.i.* Day 2 p.i. Day 3 p.i. Day 4 p.i. Day 5 p.i. Day 6 p.i. Day 7 p.i.* Day 8 p.i.* Day 9 p.i.* Day 10 p.i.* Day 11 p.i.* Minimum (days 2–6) Maximum (days 2–6) Day of max. temperature Rise in temperature from day 2 to day 3 (mC) Difference between max. and min. temperature (days 2–6) (mC) Days with temperature  40n5 mC (days 2–6) Neurological symptoms† Day of exitus

Difference between Max. max. and min. in SD

n

Mean

SD

Min.

73 73 89 89 89 88 65 49 29 28 26 26 65 65 65 89

39n04 39n15 39n50 40n59 40n76 40n75 40n82 40n16 39n25 39n09 39n04 38n97 39n45 41n16 4n65 1n09

0n36 0n35 0n53 0n54 0n48 0n52 0n66 0n67 0n63 0n40 0n27 0n29 0n50 0n43 1n14 0n65

38n5 38n6 38n5 39 39n4 39n1 38n7 38n7 38n5 38n4 38n6 38n8 38n5 39n7 3 k0n6

39n8 40n1 41n3 41n5 41n8 41n9 42n9 41n3 40n7 39n7 39n6 39n3 40n8 42n9 7 2n2

3n57 4n24 5n27 4n65 4n98 5n39 6n32 3n86 3n49 3n22 3n65 1n73 4n62 7n51 3n51 4n31

65

1n71

0n57

0n6

3n9

5n77

65

2n92

1n17

0

5

4n27

89 63

0n71 6n40

0n46 0n94

0 4

1 9

2n19 5n30

* Trait not used for QTL mapping for reasons of animal numbers. † Proportion of animals with symptoms.

offspring of these founder breeds, indicating dominant inheritance. These differences between founder breeds motivated a QTL-mapping experiment in an F 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\nonappearance 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 F 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\IFNBGJ

G. Reiner and others

Table 3. QTLs with effects on clinical signs after intranasal challenge of F2 pigs Animals were challenged with 10& p.f.u. of PrV strain NIA-3 at the age of 12 weeks. Significance thresholds : 5 % genome-wide, F  8n8 ; 10 % genome-wide, F  7n9 ; 5 % chromosome-wide, F  5n6. QTL Trait Temperature ( mC) : Day 2 p.i. Day 2 p.i. Day 4 p.i. Day 6 p.i. Day 6 p.i. Maximum (days 2–6) Maximum (days 2–6) Difference between max. and min. (days 2–6) Rise from day 2 to day 3 Day with max. temperature Day with max. temperature Neurological symptoms Neurological symptoms Neurological symptoms Day of exitus Day of exitus Day of exitus

SSC

Position (cM)

F value

Percentage of F2 variance

Additive effectpSD

Dominance effectpSD

11 17 11 4 2 2 11 8 9 10 16 9 6 5 6 9 13

64n8 56n3 34n0 150n6 106n2 106n2 55n8 124n2 129n1 136n8 35n0 142n1 22n0 46n3 19n0 144n1 41n0

9n4 5n5 6n3 6n6 5n7 6n9 5n5 6n5 5n4 8n2 5n7 8n9 7n2 7n1 6n7 6n4 5n7

19n4 11n5 13n0 13n8 16n2 14n1 11n0 13n6 9n3 16n4 9n8 17n9 14n7 14n6 13n5 13n0 11n4

k0n20p0n11 k0n13p0n11 k0n17p0n10 0n35p0n10 0n27p0n12 0n12p0n06 k0n08p0n08 0n36p0n10 k0n13p0n13 k0n11p0n16 k0n61p0n27 0n22p0n08 0n34p0n11 k0n16p0n12 k1n54p0n42 k0n72p0n32 1n33p0n42

k0n86p0n21 0n62p0n20 k0n55p0n18 0n20p0n16 0n47p0n16 0n32p0n09 k0n40p0n13 k0n09p0n19 k0n74p0n25 0n93p0n23 k0n92p0n55 0n48p0n15 0n42p0n21 k0n44p0n21 0n51p0n82 k1n54p0n55 k0n21p0n62

QTL positions Body temperature; P < 0·05* Body temperature; P < 0·10** Neurological symptoms; P < 0·05* Neurological symptoms; P < 0·10** Significance : : chromosome - wide : genome - wide 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.

BHA

QTLs for PrV resistance/susceptibility

*

80

100

120

cM

140

Sw749 -

60

Sw2093 -

40

Swr2074 APOA1 -

20

Swr1848 -

0

Sw983 -

1 0

*** **

S0024 -

F

γ 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-γ locus (SSC5) and the locus for an interferon receptor (SSC13). Thus, more specific research should include the candidate genes (PRR1, PRR2, IFN-γ, 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.

SSC9

10 9 8 7 6 5 4 3 2

SSC6

8 7 6

F

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.

*

5 4 3 2

References 60

80

100

140

120

160

Baskerville, A. (1981). Aujeszky’s disease : recent advances and current problems. New Zealand Veterinary Journal 29, 183–185. Baskerville, A., McFerran, J. B. & Dow, C. (1973). Aujeszky’s disease in pigs. Veterinary Bulletin 43, 465–480. Churchill, G. A. & Doerge, R. W. (1994). Empirical threshold values for quantitative trait mapping. Genetics 138, 963–971.

cM

Sw2052 -

40

Sw824 -

20

Swr492 -

0

Sw1329 -

0

S0087 -

1

SSC5

8 7 6

*

5

F

Eberle, F., Dubreuil, P., Mattei, M. G., Devilard, E. & Lopez, M. (1995).

The human PRR2 gene, related to the human poliovirus receptor gene (PVR), is the true homolog of the murine MPH gene. Gene 159, 267–272.

4 3 2

Favoreel, H. W., Nauwynck, H. J., Halewyck, H. M., Van Oostveldt, P., Mettenleiter, T. C. & Pensaert, M. B. (1999). Antibody-induced

1 60

80

INFG -

100

120

140

160

endocytosis of viral glycoproteins and major histocompatibility complex class I on pseudorabies virus-infected monocytes. Journal of General Virology 80, 1283–1291.

cM

180

Sw378 -

40

Sw152 -

20

Sw491 -

0 Sw413 -

0

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 0n05 genome-wide (***), P 0n10 genome-wide (**) and P 0n05 chromosome-wide (*) levels.

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receptor-related protein 1 and poliovirus receptor. Science 280, 1618–1620. Green, P., Falls, K. & Crooks, S. (1990). Documentation for Crimap version 2.4. St Louis : Washington University School of Medicine. Polykopie. Grob, P., Schijns, V. E. C. J., Van den Broek, M. F., Cox, S. P. J., Ackermann, M. & Suter, M. (1999). Role of the individual interferon

Table 4. Multiple analysis of the effects of QTLs on SSC9, SSC6 and SSC5 on appearance/non-appearance of neurological signs Animals were challenged intranasally with 10& p.f.u. PrV strain NIA-3 at the age of 12 weeks. %s F , Percentage of F variance. # # Single analysis

Multiple analysis

QTL

r

%s F2

P

r

%s F2

P

SSC9 SSC6 SSC5

0n45 0n40 0n38

20 16 15

0n0003 0n0021 0n0037

0n56 0n59

31 35

0n000

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BHC

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Received 9 August 2001 ; Accepted 18 September 2001

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