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We screened phage display libraries of porcine reproductive and respiratory syndrome virus (PRRSV) protein fragments with sera from experimentally infected pigs to identify linear B-cell epitopes that are commonly recognized during infection in vivo. We identified 10 linear epitope sites (ES) 11 to 53 amino acids in length. In the replicase polyprotein, a total of eight ES were identified, six of which localized to the Nsp2 replicase polyprotein processing end product. In the structural proteins, a total of two ES were identified, in the ORF3 and ORF4 minor envelope glycoproteins. The ORF4 ES was previously identified by monoclonal antibody mapping (J. J. M. Meulenberg, A. P. van Nieuwstadt, A. van Essen-Zandenbergen, and J. P. M. Langeveld, J. Virol. 71:6061-6067, 1997), but its immunogenicity had not been examined in pigs. We found that six experimentally PRRSV-infected pigs consistently had very high antibody titers against the ORF4 ES. In some animals, sera diluted 1:62,500 still gave weak positive enzyme immunoassay reactivity against the ORF4 ES. This hitherto unrecognized immunodominance likely caused phages displaying the ORF4 ES to outcompete phages displaying other ES during library screening with porcine sera and accounted for our failure to identify more than two ES in the structural genes of PRRSV. Genetic analysis showed that variable ES were also the most immunogenic in vivo. Serological analysis indicated differences in the immunoglobulin A responses between short-term and longer-term viremic pigs towards some ES. The implications of these findings for PRRSV diagnostics and immunopathogenesis are discussed.Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently emerged pathogen of domesticated swine. The virus, which belongs to the Arteriviridae family, has a 15-kb positive-sense, single-stranded RNA genome. PRRSV encodes an approximately 4,000-amino-acid large replicase polyprotein (open reading frame [ORF] 1a and 1b) and six structural proteins of 130 to 265 amino acids (ORFs 2 to 7) (reviewed in references 6, 36, and 45). The replicase polyprotein is processed by autoproteolytic cleavage into nonstructural protein fragments (Nsps). The replicase polyprotein processing cascade has recently been reviewed by Ziebuhr et al. (45), and the Nsp and protease domain nomenclature suggested by Ziebuhr et al. is used throughout this article. Two main PRRSV genotypes exist, the American (US) and European (EU) types, which are only approximately 60% identical at the nucleotide level. For reasons currently not understood, these two distantly related PRRSV types emerged virtually simultaneously on their respective continents in the late 1980s. Since then, intermingling of the genotypes has occurred through the use of a live, US-type PRRSV vaccine in Europe (2, 18).PRRSV infection poses a challenge to current serodiagnostic and vaccination strategies. Although live PRRSV vaccines provide protection against homologous challenge, the genetic diversity of field PRRSV isolates is very high, and v...

ABSTRACT.Purpose: To investigate incidence, clinicopathological features and prognosis of BRAF-mutated conjunctival melanoma in Denmark. Furthermore, to determine BRAF mutations in paired premalignant lesions and evaluate immunohistochemical BRAF V600E oncoprotein detection. Methods:Data from 139 patients with conjunctival melanoma were collected. Archived conjunctival melanoma samples and premalignant lesions were analysed for BRAF mutations using droplet digital polymerase chain reaction (PCR). Results were associated with clinicopathological features and compared with BRAF V600E oncoprotein stainings.Results: The overall incidence of conjunctival melanoma (0.5 cases/1 000 000/year) increased during the study period with 0.13 cases/1 000 000/10 years. The increase comprised a higher proportion of patients aged >65 years, epibulbar tumours and tumours developed from a primary acquired melanosis with atypia. BRAF mutations were identified in 39 of 111 (35%) cases. The rate ratio of BRAF-mutated versus BRAFwild-type melanoma did not change over time. BRAF mutations were associated with T1 stage (p = 0.007), young age (p = 0.001), male gender (p = 0.02), sun-exposed location (p = 0.01), mixed/non-pigmented tumour colour (p = 0.02) and nevus origin (p = 0.005), but did not associate with prognosis. BRAF status in conjunctival melanoma and paired premalignant lesions corresponded in 19 of 20 cases. Immunohistochemistry detected BRAF V600E mutations with a sensitivity of 0.94 and a specificity of 1.00 in newer conjunctival melanoma samples (2000-2012, n = 47). Conclusion:The incidence of conjunctival melanoma increased in Denmark over 50 years. The proportion of BRAF-mutated conjunctival melanoma was constant. BRAF mutations were identified as early events in conjunctival melanoma, associated with a distinct clinicopathological profile, similar to BRAF-mutated cutaneous melanoma. Immunohistochemical detection of BRAF can be used to assess BRAF V600E mutations.