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•PRRSV-specific T cell responses show highly variable kinetics among individuals in virulent PRRSV-infected pigs.•Cell-mediated immunity against PRRSV is broadly cross reactive among type 2 isolates of varying genetic distance. This cross reactivity does not extend to type 1 isolates.•Total antibody responses are cross reactive, instead neutralizing antibody titers are specific for the challenge isolate.•Homologous T cell responses show a positive association with homologous neutralizing antibody titers. Because porcine reproductive and respiratory syndrome virus (PRRSV) exhibits extensive genetic variation among field isolates, characterizing the extent of cross reactivity of immune responses, and most importantly cell-mediated immunity (CMI), could help in the development of broadly cross-protective vaccines. We infected 12 PRRSV-naïve pigs with PRRSV strain FL12 and determined the number of interferon (IFN)-γ secreting cells (SC) by ELISpot assay using ten type 2 and one type 1 PRRSV isolates as recall antigens. The number of IFN-γ SC was extremely variable among animals, and with exceptions, late to appear. Cross reactivity of IFN-γ SC among type 2 isolates was broad, and we found no evidence of an association between increased genetic distance among isolates and the intensity of the CMI response. Comparable to IFN-γ SC, total antibodies evaluated by indirect immunofluorescence assay (IFA) were cross reactive, however, neutralizing antibody titers could only be detected against the strain used for infection. Finally, we observed a moderate association between homologous IFN-γ SC and neutralizing antibodies.

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most devastating swine diseases worldwide, resulting in immense economic losses. PRRS virus (PRRSV) is divided into two major genotypes, European (type 1) and the North American (type 2). Type 1 PRRSV have recently emerged in Fujian province (South China), and this might have a significant impact on the Chinese pig industry. From 2013 to 2014, two type 1 PRRSV strains, named FJEU13 and FJQEU14, were isolated from piglets and sows with respiratory problems and reproductive disorders in Fujian province. The full genome length of the two isolates was 14,869-15,062 nucleotides (nt), excluding the poly(A) tail. These isolates shared 86.0-89.9% sequence identity with the prototypic strains Lelystad virus (LV) and 82.8-92% with Chinese type 1 PRRSV strains, but only 59.9-60.1% with the North American reference strain VR-2332. However, they were 82.9% identical to each other. Nonstructural protein 2 (Nsp2) and ORF3-ORF5 were the most variable regions when compared to other type 1 PRRSV strains. Nsp2 and ORF3 contained multiple discontinuous deletions and a 204-bp deletion in NSP2 in isolate FJQEU14, which has never been described in other Chinese type 1 PRRSV strains. All of these results might be useful for understanding the epidemic status of type 1 PRRSV in China.

•Five PRRSV vaccines have been tested for the efficacy to NADC30-like PRRSV challenge.•Vaccinated pigs had improved clinical manifestations compared to unvaccinated ones.•However, vaccinated pigs developed similar viremia and suffered pathological lesions.•PRRSV vaccines could not provide complete protection to NADC30-like PRRSV infection. NADC30-like PRRSV has been recently reported and became endemic in vaccinated pig herds in China. The outbreaks of disease in vaccinated pigs indicated the inefficacy of commercial PRRSV vaccines. In this study, five commercial PRRSV vaccines that have been widely used in China were used to evaluate the efficacy to a NADC30-like PRRSV infection. The vaccinated pigs were challenged with HNjz15, a NADC30-like PRRSV at 28days post vaccination. Compared to unvaccinated pigs, the vaccinated pigs clinically shortened the period of fever with less pig numbers of clinical manifestations and had improved body weight gain at the end of the study. However, the vaccinated pigs developed viremia with similar kinetics and suffered pathological lesions in lung and lymphoid tissues as the unvaccinated pigs. The virus load in tonsil, lung and lymph nodes detected by immunohistochemistry staining in vaccinated pigs was also similar to that in unvaccinated pigs which indicated the inability of vaccination to eradicate the virus from tissues of vaccinated pigs. Therefore, the above results suggested current commercial PRRSV vaccines could not provide complete protection to the NADC30-like PRRSV infection.

► PRRS is a devastating disease of pigs despite availability of vaccines since 1994. ► Mechanisms of protective immunity are poorly understood. ► Correlates of immune protection are not known. ► Viral diversity complicates disease control and research interpretations. ► Understanding the limitations will help improve research efforts. Vaccination is the principal means used to control and treat porcine reproductive and respiratory syndrome virus (PRRSV) infection. An array of PRRS vaccine products is available in various regions of the world. However, despite extensive efforts, little progress has been made to improve efficacy since the first introduction of a live, attenuated vaccine in 1994 in the USA. Key limitations include: (a) uncertainty about the viral targets of protective immunity that prevents a research focus on individual viral structures and proteins, and frustrates efforts to design novel vaccines; (b) inability to establish clear immunological correlates of protection that requires laborious in vivo challenge models for evaluation of protection against challenge; and (c) the great genetic diversity of PRRSV which requires that challenge experiments be interpreted cautiously since it is not possible to predict how immunological protection against one isolate will translate to broadly cross-protective immunity. Economically significant levels of cross-protection that are provided to a variety of field isolates still cannot assure that effective protection will be conferred to isolates that might emerge in the future. In addition to these substantial barriers to new PRRSV vaccine development, there are enormous gaps in our understanding of porcine immunological mechanisms and processes that provide immunity to PRRSV infection and memory responses for long-term protection. Despite these impediments, we should be confident that progress will be made. Sequencing of the swine genome is providing a rich source of primary knowledge of gene structure and transcriptional regulation that is certain to reveal important insights about the mechanisms of anti-PRRSV immunity, and continued efforts to unravel the details of the interaction of PRRSV with pigs will lead to new insights that overcome the current limitations in the field.

Current porcine reproductive and respiratory syndrome virus (PRRSV) vaccines sometimes fail to provide adequate immunity to protect pigs from PRRSV-induced disease. This may be due to antigenic differences among PRRSV strains. Rapid production of attenuated farm-specific homologous vaccines is a feasible alternative to commercial vaccines. In this study, attenuation and efficacy of a codon-pair de-optimized candidate vaccine generated by synthetic attenuated virus engineering approach (SAVE5) were tested in a conventional growing pig model. Forty pigs were vaccinated intranasally or intramuscularly with SAVE5 at day 0 (D0). The remaining 28 pigs were sham-vaccinated with saline. At D42, 30 vaccinated and 19 sham-vaccinated pigs were challenged with the homologous PRRSV strain VR2385. The experiment was terminated at D54. The SAVE5 virus was effectively attenuated as evidenced by a low magnitude of SAVE5 viremia for 1–5 consecutive weeks in 35.9% (14/39) of the vaccinated pigs, lack of detectable nasal SAVE5 shedding and failure to transmit the vaccine virus from pig to pig. By D42, all vaccinated pigs with detectable SAVE5 viremia also had detectable anti-PRRSV IgG. Anti-IgG positive vaccinated pigs were protected from subsequent VR2385 challenge as evidenced by lack of VR2385 viremia and nasal shedding, significantly reduced macroscopic and microscopic lung lesions and significantly reduced amount of PRRSV antigen in lungs compared to the non-vaccinated VR2385-challenged positive control pigs. The nasal vaccination route appeared to be more effective in inducing protective immunity in a larger number of pigs compared to the intramuscular route. Vaccinated pigs without detectable SAVE5 viremia did not seroconvert and were fully susceptible to VR2385 challenge. Under the study conditions, the SAVE approach was successful in attenuating PRRSV strain VR2385 and protected against homologous virus challenge. Virus dosage likely needs to be adjusted to induce replication and protection in a higher percentage of vaccinated pigs.

•Porcine reproductive and respiratory syndrome (PRRS) is a chronic and economically devastating disease of pigs since the late 1980s.•Although modified live-attenuated PRRSV (PRRSV-MLV) vaccines have been used since 1995, control of PRRS globally is still a challenge.•PRRSV-MLV provides incomplete protection against existing and emerging genetically variant field isolates.•Promising approaches to improve PRRSV-MLV efficacy, are under experimental study.•This review highlights the current status and future directions of infectious PRRSV vaccine. Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) was reported in the late 1980s. PRRS still is a huge economic concern to the global pig industry with a current annual loss estimated at one billion US dollars in North America alone. It has been 20 years since the first modified live-attenuated PRRSV vaccine (PRRSV-MLV) became commercially available. PRRSV-MLVs provide homologous protection and help in reducing shedding of heterologous viruses, but they do not completely protect pigs against heterologous field strains. There have been many advances in understanding the biology and ecology of PRRSV; however, the complexities of virus-host interaction and PRRSV vaccinology are not yet completely understood leaving a significant gap for improving breadth of immunity against diverse PRRS isolates. This review provides insights on immunization efforts using infectious PRRSV-based vaccines since the 1990s, beginning with live PRRSV immunization, development and commercialization of PRRSV-MLV, and strategies to overcome the deficiencies of PRRSV-MLV through use of replicating viral vectors expressing multiple PRRSV membrane proteins. Finally, powerful reverse genetics systems (infectious cDNA clones) generated from more than 20 PRRSV isolates of both genotypes 1 and 2 viruses have provided a great resource for exploring many innovative strategies to improve the safety and cross-protective efficacy of live PRRSV vaccines. Examples include vaccines with diminished ability to down-regulate the immune system, positive and negative marker vaccines, multivalent vaccines incorporating antigens from other porcine pathogens, vaccines that carry their own cytokine adjuvants, and chimeric vaccine viruses with the potential for broad cross-protection against heterologous strains. To combat this devastating pig disease in the future, evaluation and commercialization of such improved live PRRSV vaccines is a shared goal among PRRSV researchers, pork producers and biologics companies.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a threat for the pig industry. Vaccines have been developed, but these failed to provide sustainable disease control, in particular against genetically unrelated strains. Here we give an overview of current knowledge and gaps in our knowledge that may be relevant for the development of a future generation of more effective vaccines. PRRSV replicates in cells of the monocyte/macrophage lineage, induces apoptosis and necrosis, interferes with the induction of a proinflammatory response, only slowly induces a specific antiviral response, and may cause persistent infections. The virus appears to use several evasion strategies to circumvent both innate and acquired immunity, including interference with antigen presentation, antibody-mediated enhancement, reduced cell surface expression of viral proteins, and shielding of neutralizing epitopes. In particular the downregulation of type I interferon-α production appears to interfere with the induction of acquired immunity. Current vaccines are ineffective because they suffer both from the immune evasion strategies of the virus and the antigenic heterogeneity of field strains. Future vaccines therefore must “uncouple” the immune evasion and apoptogenic/necrotic properties of the virus from its immunogenic properties, and they should induce a broad immune response covering the plasticity of its major antigenic sites. Alternatively, the composition of the vaccine should be changed regularly to reflect presently and locally circulating strains. Preferably new vaccines should also allow discriminating infected from vaccinated pigs to support a virus elimination strategy. Challenges in vaccine development are the incompletely known mechanisms of immune evasion and immunity, lack of knowledge of viral sequences that are responsible for the pathogenic and immunosuppressive properties of the virus, lack of knowledge of the forces that drive antigenic heterogeneity and its consequences for immunogenicity, and a viral genome that is relatively intolerant for subtle changes at functional sites.

The pathogenicity of HNjz15, an NADC30-like strain of porcine reproductive and respiratory syndrome virus (PRRSV), was investigated and compared to that of a highly pathogenic PRRSV JAX1 strain. Six-week-old pigs infected with each virus showed typical clinical symptoms, including high fever and respiratory disorders. Pigs infected with JXA1 had more-severe clinical manifestations than pigs infected with HNjz15. HNjz15 replicated in vivo with kinetics similar to those of JXA1 but induced a lower level of PRRSV-specific antibody at the beginning of virus infection. Histopathologically, JXA1 infection led to more-severe lung lesions and broader organ tropism than HNjz15 did. Different from what was observed with the previously reported NADC30-like PRRSV JL580 strain, all HNjz15-infected pigs survived until the end of the study. All of these results indicated that NADC30-like PRRSV HNjz15 is virulent to pigs but is less pathogenic than the JXA1 and JL580 PRRSV strains.

Porcine reproductive and respiratory syndrome virus (PRRSV), an important pathogen in the swine industry, is a genetically highly diverse RNA virus. However, the phylogenetic and genomic recombination properties of this virus are not yet fully understood. In this study, we performed an integrated analysis of all available whole-genome sequences of type 2 PRRSV (n = 901) to reveal its evolutionary dynamics. The results showed that there were three distinct phylogenetic lineages of PRRSV in their distribution patterns. We identified that sublineage 2.7 (L2.7), associated with a NADC30 cluster, had the highest substitution rate and higher viral genetic diversity, and inter-lineage recombination is observed more frequently in L2.7 PRRSV compared to other sublineages. Most inter-lineage recombination events detected are observed between L2.7 PRRSVs (as major parents) and L3.4 (a JXA1-R-related cluster)/L3.7 (a WUH3-related cluster) PRRSVs (as minor parents). Moreover, the recombination hotspots are located in the structural protein gene ORF2 and ORF4, or in the non-structural protein gene nsp7. In addition, a GM2-related cluster, L3.2, shows inconsistent recombination modes compared to those of L2.7, suggesting that it may have undergone extensive and unique recombination in their evolutionary history. We also identified several amino acids under positive selection in GP2, GP4 and GP5, the major glycoproteins of PRRSV, showing the driving force behind adaptive evolution. Taken together, our results provide new insights into the evolutionary dynamics of PPRSV that contribute to our understanding of the critical factors involved in its evolution and guide future efforts to develop effective preventive measures against PRRSV.

The nucleocapsid protein of the European genotype of porcine reproductive and respiratory syndrome virus (type 1, PRRSV-1) exhibited extensive size polymorphism (124-130 amino acids), correlating with phylogenetic grouping of ORF7 as well as ORF5 nucleotide sequences, thereby validating ORF7 size as an independent PRRSV-1 subtype marker. Based on new sequence information from the Russian Federation, we propose division of European genotype PRRSV-1 into 3 subtypes: a pan-European subtype 1 and East European subtypes 2 and 3, with nucleocapsid protein sizes of 128, 125 and 124 amino acids, respectively. The genetic differences between European genotype PRRSV subtypes affected diagnostic RT-PCR primer binding sites. Using Escherichia coli-expressed ORF7 protein, we confirmed that even the relatively closely related PRRSV subtypes 2 and 3 were antigenically different. Finally, the isoelectric point (pI) correlated with the nucleocapsid protein size for European genotype PRRSV subtypes, suggesting subtype-specific compensatory structural changes associated with subtype-specific ORF7 sizes. Thus, the new ORF7-based subtype division of PRRSV-1 proposed here is biologically meaningful and practically relevant.