Ovine progressive pneumonia provirus levels are unaffected by the prion 171R allele in an Idaho sheep flock

Selective breeding of sheep for arginine (R) at prion gene (PRNP) codon 171 confers resistance to classical scrapie. However, other effects of 171R selection are uncertain. Ovine progressive pneumonia/Maedi-Visna virus (OPPV) may infect up to 66% of a flock thus any affect of 171R selection on OPPV susceptibility or disease progression could have major impact on the sheep industry. Hypotheses that the PRNP 171R allele is 1) associated with the presence of OPPV provirus and 2) associated with higher provirus levels were tested in an Idaho ewe flock. OPPV provirus was found in 226 of 358 ewes by quantitative PCR. The frequency of ewes with detectable provirus did not differ significantly among the 171QQ, 171QR, and 171RR genotypes (p > 0.05). Also, OPPV provirus levels in infected ewes were not significantly different among codon 171 genotypes (p > 0.05). These results show that, in the flock examined, the presence of OPPV provirus and provirus levels are not related to the PRNP 171R allele. Therefore, a genetic approach to scrapie control is not expected to increase or decrease the number of OPPV infected sheep or the progression of disease. This study provides further support to the adoption of PRNP 171R selection as a scrapie control measure.

The PRNP 171Q allele predominates in US sheep whereas the 171R allele and 171RR genotype are less common (the latter two occur at a frequency of about 37% and 16%, respectively [20]). Selective breeding for the 171R minor allele to produce animals with the 171QR or 171RR genotypes is sometimes used as a Scrapie control measure, however the functional consequences of 171R selection on other traits is uncertain. Genetic selection may have unexpected positive or negative effects as individual genes may have multiple biological roles (pleiotropy) or may be linked to other genes that impact overall biological functions. Uncertainty regarding PRNP selection effects (beyond Scrapie resistance) has led to investigation of multiple ovine traits related to reproduction, milk, meat, fiber and genetic diversity. However, PRNP selection effects on disease susceptibility (besides Scrapie) has only been studied for  [32]. In this study, the following two hypotheses were tested in an Idaho ewe flock: 1) the PRNP codon 171R allele is associated with the presence of OPPV provirus and 2) the PRNP 171R allele is associated with higher OPPV provirus levels. This study will help guide producer decisions and it provides information for future prion-retrovirus co-infection studies and advances knowledge of whether PRNP selection affects other infectious diseases.

Nucleic acid extraction
Peripheral blood leukocytes (PBL) were isolated from whole blood as described [23]. Genomic DNA was extracted from PBL using a commercial kit (Gentra, Minneapolis, Minnesota).

Statistical analyses
Two types of genotypic comparison were made using provirus data and PRNP genotype, with a minimum PRNP allele frequency of 10% required for analysis. Association between PRNP genotype and presence or absence of OPPV provirus was tested using logistic regression models from the logistic procedure of SAS v9.1 (SAS Institute, Cary, NC). Association between PRNP genotype and the level of logarithm (base 10)-transformed provirus in OPPV positive animals was tested using the glm procedure in SAS v9.1. In each case the association model included breed as a categorical predictor, age as a linear covariate, the interaction between breed and age, and the PRNP genotype of interest. Adjusted odds ratios and 95% confidence interval were calculated for the pair-wise comparison of the frequency of OPPV positive ewes in each PRNP genotype. Adjusted mean log-transformed provirus levels were reported with 95% confidence intervals.
Stepdown Bonferroni p-value correction [35] was applied separately to each set of analyses.

Distribution of PRNP genotypes
The PRNP genotypes were determined as the first step in testing association with the presence of OPPV provirus and OPPV provirus levels. PRNP ORF coding variants were identified at codons 101(Q/R), 136(A/V), 141(L/F), 143 (H/R), 154 (R/H), and 171 (Q/R) ( Table 1). Of the 358 sheep sampled, 100 (28%) were 171QQ, 179 (50%) were 171QR and 79 (22%) were 171RR, providing a representation of all three genotypes (Fig. 1, left). Examination of the 171R allele relative to the overall PRNP ORF showed that in all animals with the 171RR genotype there were no other PRNP codon variants present. Codon changes at other positions only occurred in animals that had at least one wild type 171Q allele. Of the 358 sheep, 279 (78%) were 143HH, 71 (20%) were 143HR and 8 (2%) were 143RR (Fig. 1, right). Since codons 143 and 171 had amino acid substitutions with a minor allele frequency of at least 10% they were further analyzed, except for the rare 143RR genotype. Codons 101, 136, 141, and 154 had a minor allele frequency of less than 10% and therefore these four codons were excluded from further association analysis.

Frequency of OPP provirus among PRNP genotype
The presence or absence of OPPV provirus was compared among the PRNP 171 and PRNP 143 genotypes, using a statistical model accounting for age and breed, to determine if minor alleles within those genotypes affected the number of sheep that had detectable OPPV provirus. In the flock, 226 of 358 (63.1%) sheep had detectable OPPV provirus. Over half of the ewes were positive for OPPV provirus within each PRNP 171 or 143 genotype ( Table  2). The frequency of OPPV positive animals was not significantly different between the 171QQ, QR, and RR genotypes as indicated by nominal and corrected p-values greater than 0.05 (Table 3) and equivalent odds ratios (Fig. 2). The 95% confidence intervals also indicate the range of potential effect sizes consistent with these data (Fig. 2). Also, the frequency of OPPV positive animals did not differ significantly between the 143HH and HR genotypes.

OPPV provirus levels among PRNP genotypes
The levels of OPPV provirus were compared among the PRNP 171 and PRNP 143 genotypes to determine whether particular genotypes were associated with higher or lower provirus levels once a ewe became infected. Adjusted mean log-transformed provirus levels with 95% confidence interval were equivalent among codon 171 and among codon 143 genotypes (Fig. 3). Adjusted mean logtransformed provirus levels were not significantly different among the 171QQ, QR, and RR genotypes or among the 143HH and HR genotypes in which nominal and corrected p-values were greater than 0.05 (Table 4).

Discussion
The present study was performed to determine if a PRNP 171R selection program impacts the presence or magnitude of OPPV infection. Allelic variation in PRNP could affect OPPV status if PRNP variants produce changes in PrP c function or expression level relevant to OPPV, if is possible that introduction of rams could have a different affect, however it is unlikely considering that the frequency of OPPV in rams is equivalent, or perhaps lower than OPPV frequency in ewes [36,22].
Also, provirus levels in OPPV positive animals were not related to the PRNP 171R and 143R alleles. Thus, PRNP selection should not affect progression of disease once animals become infected with OPPV. A shift of flock genetics to a greater frequency of 171QR or 171RR sheep is unlikely to accelerate shedding or transmission of OPPV. In these sheep there also was no difference in provirus levels between animals of the 143 HH and 143HR genotypes, thus there are no documented cases where PRNP genotypes impact OPPV infection.
Recent studies have shown that factors such as breed and age are important for OPPV, therefore all analyses in this study accounted for breed, age and differences in how each breed handled OPPV with age. For example, Ram-    [37]. However, all the analyses in this study accounted for age and breed in the association models so that these factors would not influence tests for association with PRNP genotype.

Number of sheep distributed among PRNP genotypes
Interactions between retrovirus' and normal or abnormal prion protein have been previously observed. The current findings do not exclude the possibility that increases in ovine PrP c or CD230 expression could alter OPPV replication as observed in a human cell line where over-expression of human PrP c thwarted HIV-1 replication [32]. OPPV replicates in mammary macrophages and microglia and transmits via ewe milk [38][39][40] and PrP Sc is found in macrophages of lymphoid follicles and microglia and transmits via ewe milk [41][42][43][44]31]  This study is one of many examining PRNP selection effects. The PRNP 171RR genotype has no apparent effect on reproductive performance [45,46], ovulation rates and litter sizes [47], and only the Suffolk breed has lower lamb weaning weights [48]. Milk production and quality is not effected in Churra [49], East Friesian milk sheep [46] or Sardinian sheep and there are no significant changes in udder morphology [50]. Carcass and wool quality are not impaired [46,21] and 171R may positively affect average daily gain [51]. 171R has no effect on Salmonella resistance [21]. Finally, pedigree examination in Laxta Black Facedtype Navarra sheep showed no overall negative effect [52].
The present study taken together with previous investigations indicate that the correlated responses to PRNP 171R selection should be minimal. In total, ten different studies examining reproduction, meat, milk, fiber and infectious disease traits in a dozen different breeds found no overt negative effect from the PRNP 171R allele or 171RR genotype. Additional studies may supplement present and previous results by examining other breeds, genotypes, retrovirus strains, diseases, environmental or management conditions, or production traits. This investigation of a flock with endemic OPPV shows that the frequency of OPPV infection and level of OPPV provirus loads are not Odds ratio and 95% confidence interval for effect of PRNP genotype upon frequency of OPPV positive animals   Adjusted mean log10 provirus levels and 95% confidence interval among PRNP genotypes used for statistical comparison