In a recent publication, it was demonstrated that the PRRSV-1 subtype 1 vaccine strain modified live virus (MLV)-DV (GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”KJ127878″,”term_id”:”575379611″,”term_text”:”KJ127878″KJ127878), which is closely related to the old prototype virus LV (98.4% identity of full genome sequences) (GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”M96262″,”term_id”:”11125727″,”term_text”:”M96262″M96262), was able to give partial clinical and virologic protection against PRRSV-1 subtype 3 strain Lena [3]. against strains used for the first inoculation. Our results indicate that inoculation with subtype 1 PRRSV strains can partially protect against antigenically divergent subtype 3 strains. The lower protection level elicited by recently isolated subtype 1 PRRSV strains may impair the outcome NUN82647 of the spatial expansion of subtype 3 strains from East Europe to West Europe. Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent in most swine farms worldwide, and is a major cause of economic losses and animal suffering. Current genetic analysis of a number of European genotype PRRSV-1 strains reveals the existence of four different subtypes [1]. In Europe, a geographical demarcation exists between areas of low (Western and Central Europe) and high (Eastern Europe) PRRSV-1 diversity [1]. New Belgian PRRSV-1 variants still belong to subtype 1, but genetic changes have led to an CD340 increase in virulence and pathogenicity [2]. This drift has resulted in further economic losses in the swine industry in 2013 and 2014. Stability of the spatial distribution of different PRRSV subtypes in Europe allows us to continue using the term East European subtypes for subtypes 2, 3 and 4. However, there is a potential risk that East European subtypes, which are genetically and antigenically distinct from Pan-European subtype 1 viruses and are even more virulent/pathogenic, could emerge in Western and Central Europe, leading to a real catastrophe [1, 3, 4]. The role of existing herd immunity in keeping subtypes 2, 3 and 4 out of Western NUN82647 and Central Europe is of great interest. Active immunization is currently the only widely available and approved way to control PRRS-related problems in swine herds worldwide. The existence of different PRRSV subtypes requires vaccines that induce a strong cross-protective immune response. In a recent publication, it was demonstrated that the PRRSV-1 subtype 1 vaccine strain modified live virus (MLV)-DV (GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”KJ127878″,”term_id”:”575379611″,”term_text”:”KJ127878″KJ127878), which is closely related to the old prototype virus LV (98.4% identity of full genome sequences) (GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”M96262″,”term_id”:”11125727″,”term_text”:”M96262″M96262), was able to give partial clinical and virologic protection against PRRSV-1 subtype 3 strain Lena [3]. With the appearance of genetically more distant PRRSV strains, the question raised as to what degree these strains induce a protective immunity against PRRSV Lena. In the present study, the level of protection against European PRRSV subtype 3 strain Lena was examined in animals immunized with one old and two recent subtype 1 PRRSV-1 strains. Materials and methods Animals, experimental design and inoculation viruses Twenty conventional pigs were obtained from a PRRS-negative farm. All animals were housed in separate stables in a biosafety level 2 (BSL2) facility at the Faculty of Veterinary Medicine, Ghent University, Belgium. No relevant pathogens (PRRSV, SIV, PCV2) were detected in the animals. Pigs were randomly divided into four groups with five animals per group (07V063, 13V091, 13V117, and a mock-inoculated control group). Two consecutive inoculations were performed in this experiment. Pigs were 11?weeks old at NUN82647 the first inoculation and 18?weeks old at the second inoculation. Inoculations were performed intranasally using a 2??105 tissue culture infectious dose (TCID) with a 50% end point (TCID50) PRRSV. The 07V063 group was inoculated with the third NUN82647 passage of 07V063 strain: the 13V091 group with the third passage of 13V091 strain; and the 13V117 group with the second passage of 13V117 strain; all propagated in porcine alveolar macrophages (PAMs). These subtype 1 PRRSV-1 strains were isolated from different Belgian farms in 2007 (07V063) and 2013 (13V091 and 13V117). Subtype 1 PRRSV 07V063 strain is a mildly pathogenic Belgian strain [2, 4]. Newly isolated strains originated from NUN82647 farms with animals experiencing endemic respiratory disorders in nursery pigs (13V091 and 13V117) [2]. The control group was inoculated intranasally using 1?mL phosphate-buffered saline (PBS) per nostril. Seven weeks after the first inoculation all four groups were inoculated with the PRRSV Lena strain (forth passage propagated in PAMs). Lena is a highly pathogenic East European subtype 3 PRRSV strain isolated from aborted fetuses on a Belarusian farm [3C5]. Individual sterile syringes and plastic cannulas (Jorgenson Labs J12) were used for intranasal virus inoculation. The sequences of 07V063, 13V091, 13V117 and Lena were downloaded from GenBank (GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”GU737264″,”term_id”:”339409383″,”term_text”:”GU737264″GU737264, “type”:”entrez-nucleotide”,”attrs”:”text”:”KT159248″,”term_id”:”952985705″,”term_text”:”KT159248″KT159248, “type”:”entrez-nucleotide”,”attrs”:”text”:”KT159249″,”term_id”:”952985716″,”term_text”:”KT159249″KT159249, “type”:”entrez-nucleotide”,”attrs”:”text”:”JF802085″,”term_id”:”349502023″,”term_text”:”JF802085″JF802085). Alignment and phylogenetic analysis were performed using the Mobyle@Pasteur web bioinformatics framework [6]. Clinical and pathological examinations.