Immunogenicity of the highly pathogenic porcine reproductive and respiratory syndrome virus GP5 protein encoded by a synthetic ORF5 gene

Since May 2006, a highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV), which causes continuous high fever and a high proportion of deaths in vaccinated pigs of all ages, has emerged and prevailed in Mainland China. Huge efforts should be made towards the development of an efficient vaccine against the highly pathogenic PRRSV. Although the ORF5-encoded GP5 is the most important immunogenic protein, accumulating evidences have demonstrated that incomplete protection conferred by GP5-based vaccines. The inability to induce robust protective immunity has been postulated to be associated with the presence of a non-neutralizing decoy epitope and heavy glycosylation in close to its neutralizing epitope. In this study, a synthetic ORF5 gene (SynORF5) was engineered with the codon usage optimized for mammalian cell expression based on the native ORF5 gene of highly pathogenic PRRSV strain WUH3. Additional modifications, i.e., inserting a Pan DR T-helper cell epitope (PADRE) between the neutralizing epitope and the non-neutralizing decoy epitope, and mutating four potential N-glycosylation sites (N30, N34, N35 and N51) were also included in the synthetic ORF5 gene. The immunogenicity of the SynORF5-encoded GP5 was evaluated by DNA vaccination in mice and piglets. Results showed that significantly enhanced GP5-specific ELISA antibody, PRRSV-specific neutralizing antibody, IFN-γ level, as well as lymphocyte proliferation response, could be induced in mice and piglets immunized with DNA construct encoding the modified GP5 than those received DNA vaccine expressing the native GP5. The enhanced immunogenicity of the modified GP5 will be useful to facilitate the development of efficient vaccines against the highly pathogenic PRRSV in the future.     

DNA vaccines co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV) display enhanced immunogenicity

The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. In the present study, three different DNA vaccine constructs, expressing GP5 alone (pCI-ORF5), M alone (pCI-ORF6) or GP5 and M proteins simultaneously (pCI-ORF5/ORF6), were constructed. In vitro, the co-expressed GP5 and M proteins could form heterodimeric complexes in transfected cells and heterodimerization altered the subcellular localization of GP5. The immunogenicities of these DNA vaccine constructs were firstly investigated in a mouse model. Mice inoculated with pCI-ORF5/ORF6 developed PRRSV-specific neutralizing antibodies at 6 and 8 weeks after primary immunization. However, only some mice developed low levels of neutralizing antibodies in groups immunized with pCI-ORF5 or pCI-ORF6. The highest lymphocyte proliferation responses were also observed in mice immunized with pCI-ORF5/ORF6. Interestingly, significantly enhanced GP5-specific ELISA antibody could be detected in mice immunized with pCI-ORF5/ORF6 compared to mice immunized with pCI-ORF5. The immunogenicities of pCI-ORF5/ORF6 were further evaluated in piglets (the natural host) and all immunized piglets developed neutralizing antibodies at 10 weeks after primary immunization, whereas there was no detectable neutralizing antibodies in piglets immunized with pCI-ORF5. These results indicate that the formation of GP5/M heterodimers may be involved in post-translational modification and transport of GP5 and may play an important role in immune responses against PRRSV infection. More importantly, co-expression of GP5 and M protein in heterodimers can significantly improve the potency of DNA vaccination and could be used as a strategy to develop a new generation of vaccines against PRRSV.     

Alternative codon usage of PRRS virus ORF5 gene increases eucaryotic expression of GP(5) glycoprotein and improves immune response in challenged pigs

Pigs exposed to GP(5) protein of PRRSV by means of DNA immunization develop specific neutralizing and protecting antibodies. Herein, we report on the consequences of codon bias, and on the favorable outcome of the systematic replacement of native codons of PRRSV ORF5 gene with codons chosen to reflect more closely the codon preference of highly expressed mammalian genes. Therefore, a synthetic PRRSV ORF5 gene (synORF5) was constructed in which 134 nucleotide substitutions were made in comparison to wild-type gene (wtORF5), such that 59% (119) of wild-type codons were replaced with known preferable codons in mammalian cells. In vitro expression in mammalian cells of synORF5 was considerably increased comparatively to wtORF5, following infection with tetracycline inducible replication-defective human adenoviral vectors (hAdVs). After challenge inoculation, SPF pigs vaccinated twice with recombinant hAdV/synORF5 developed earlier and higher antibody titers, including virus neutralizing antibodies to GP(5) than pigs vaccinated with hAdV/wtORF5. Data obtained from animal inoculation studies suggest direct correlation between expression levels of immunogenic structural viral proteins and immune response.     

Production and immunogenicity of chimeric virus-like particles containing porcine reproductive and respiratory syndrome virus GP5 protein

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a severe threat in swine industry and causes heavy economic losses worldwide. Currently, the available vaccines are the inactivated and attenuated virus vaccines, but the use of PRRSV in their production raises the issue of safety. We developed a chimeric virus-like particles (VLPs) vaccine candidate for PRRSV protection. The chimeric VLPs was composed of M1 protein from H1N1 influenza virus and a fusion protein, denoted as NA/GP5, containing the cytoplasmic and transmembrane domains of H1N1 virus NA protein and PRRSV GP5 protein. Vaccination of BALB/c mice with 10 μg of chimeirc VLPs by intramuscular immunization stimulated antibody responses to GP5 protein, and induced cellular immune response. The data suggested that the chimeric VLP vaccine candidate may provide a new strategy for further development of vaccines against PRRSV infection.     

Porcine reproductive and respiratory syndrome virus diversity of Eastern Canada swine herds in a large sequence dataset reveals two hypervariable regions under positive selection

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to be genetically highly variable, but knowledge of sequence diversity from Eastern Canada and its degree of genetic plasticity in or near the principal neutralizing epitope (PNE) in association with evolutionary selective pressure is limited. The purposes of our study were to investigate the extent of strain diversity, the existing glycotypes and the amino acid sites under selective evolutionary pressure in its encoded protein, GP5, for a dataset of 1301 sequences (1998-2009). This was addressed by partitioning and clustering into subgenotypes a large number of open reading frame 5 sequences from the province of Quebec and analyzing the content of these subgenotypes. The overall pairwise diversity was 12% and was comparable to what has been reported around the world. The mean diversity for sequences within subgenotypes was around 7%. No marked variations in subgenotype emergence could be observed through time. Thirty-eight GP5 glycotype patterns were observed which included a newly identified site at position N57 which was already present in 1998. These patterns possessed one to six N-glycosylation sites in total and could be located in eight different positions. No obvious grouping of glycotypes could be established in relation to subgenotypes. Positions N44 and N51 were confirmed to be fixed N-glycosylation positions, whereas other positions where found to be shifting and located in or near hypervariable regions (HVRs) 1 and 2. Both HVRs were under selective evolutionary pressure in half of all subgenotypes including vaccine-like groups. Conversely, the PNE flanked by both HVRs was well conserved among most subgenotypes demonstrating potential molecular constraint in a probable viral binding region. The analysis of this dataset increased knowledge of evolutionary change inferred from genetic data, more specifically regarding the implications of both HVRs in PRRSV diversity.     

Analysis of synonymous codon usage in porcine reproductive and respiratory syndrome virus

In this study, we calculated the relative synonymous codon usage (RSCU) values and codon usage bias (CUB) values to implement a comparative analysis of codon usage pattern of open reading frames (ORFs) which belong to the two main genotypes of porcine reproductive and respiratory syndrome virus (PRRSV). By analysis of synonymous codon usage values in each ORF of PRRSV, the optimal codons for most amino acids were all C or G-ended codons except GAU for Asp, CAU for His, UUU for Phe and CCU for Pro. The synonymous codon usage patterns in different ORFs of PRRSV were different and genetically conserved. Among them, ORF1a, ORF4, ORF5 and ORF7 could cluster these strains into the two main serotypes (EU and US). Due to mutational pressure, compositional constraint played an important role in shaping the synonymous codon usage pattern in different ORFs, and the synonymous codon usage diversity in ORFs was correlated with gene function. The degree of CUB for some particular amino acids under strong selection pressure probably served as a potential genetic marker for each ORF in PRRSV. However, gene length and translational selection in nature had no effect on the synonymous codon usage pattern in PRRSV. These conclusions could not only offer an insight into the synonymous codon usage pattern and differentiation of gene function, but also assist in understanding the discrepancy of evolution among ORFs in PRRSV.     

HSP70 fused with GP3 and GP5 of porcine reproductive and respiratory syndrome virus enhanced the immune responses and protective efficacy against virulent PRRSV challenge in pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) has been mainly responsible for the heavy economic losses in pig industry all over the world. Current vaccination strategies provide only a limited protection. In this study recombinant adenoviruses expressing GP3/GP5 of highly pathogenic PRRSV and heat shock protein 70 (HSP70) gene of Heamophilus parasuis were constructed, and the immune responses and protective efficacy against homologous challenge were examined in pigs. The results showed that all animals vaccinated with rAd-GP35 (co-expressing GP3-GP5), rAd-HS35 and rAd-HSA35 (co-expressing GP3-GP5 fused with HSP70 using different linkers), developed specific anti-PRRSV ELISA antibody and neutralizing antibody. The humoral immune responses of rAd-HS35, especially rAd-HSA35 containing 2A of FMDV between HSP70 and GP3 gene, were significantly higher than that of rAd-GP35. Moreover, the fusion of HSP70 markedly induced both IFN-γ and IL-4 in pigs’ sera. Following challenge with PRRSV, pigs inoculated with recombinant rAd-HS35 and rAd-HSA35 showed lighter clinical signs, lower viremia and less pathological lesion of lungs, as compared to those in rAd-GP35 group. Moreover, the protective efficiency induced by rAd-HSA35 was higher than that of rAd-HS35. It indicated that HSP70 fused with GP3 and GP5 of PRRSV could induce enhanced immune responses and provide protection against virulent PRRSV challenge in pigs. The recombinant adenovirus rAd-HSA35 might be an attractive candidate vaccine for the prevention and control of highly pathogenic PRRSV infections.     

Construction and immunogenicity of DNA vaccines encoding fusion protein of murine complement C3d-p28 and GP5 gene of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) has recently caused catastrophic losses in swine industry worldwide. The commercial vaccines only provide a limited protection against PRRSV infection. At present, DNA vaccine is the focus on the new vaccines. The gene fragment (p28) coding for the molecular adjuvants complement protein C3d (mC3d) from BALB/c mouse was cloned and expressed as a fusion protein for its application in the vaccine study of mice. Three potential vaccines construct units were engineered to contain two, four and six copies of mC3d-p28 coding gene linked to the GP5 gene of PRRSV and one vaccine expressing GP5 alone (pcDNA3.1-GP5) was constructed. Subsequently, the vaccines’ abilities to elicit the humoral and cellular immune responses were investigated in mice. These results showed that significantly enhanced GP5-specific ELISA antibody, GP5-specific neutralizing antibody, IFN-γ level, and IL-4 level, could be induced in mice immunized with DNA construct units encoding the pcDNA3.1-C3d-p28.n-GP5 than those received DNA vaccine expressing GP5 alone (pcDNA3.1-GP5). Analysis of the immunogenicity of different repeats of mC3d-p28 revealed that mC3d-p28 had an enhancing effect on the immunogenicity of antigens, and that six or more repeats of mC3d-p28 may be necessary for efficient enhancement of antigen specific immune responses. This approach may provide a new strategy for the development of efficient vaccines against the PRRSV for pigs in the future.     

Protective humoral immune response induced by an inactivated porcine reproductive and respiratory syndrome virus expressing the hypo-glycosylated glycoprotein 5

Porcine reproductive and respiratory syndrome (PRRS) causes significant economic losses to the swine industry worldwide. Although inactivated and live vaccines are commercially available for the control of PRRS, both types of vaccine have not always proven successful in terms of generating a protective immune response, particularly in the case of inactivated vaccines. In this study, we tested whether an inactivated vaccine could induce a humoral immune response to PRRS during a homologous challenge. Amino acid substitutions were introduced into glycoprotein (GP) 5 of the FL12 strain of the PRRS virus (PRRSV) using site-directed mutagenesis with a pFL12 infectious clone. The substitutions led to double deglycosylation in the putative glycosylation moieties on GP5. The mutant virus was subsequently inactivated with binary ethylenimine. The efficacy of the inactivated mutant virus was compared with that of the inactivated wild-type PRRSV. Only the inactivated mutant PRRSV induced serum neutralizing antibodies at six weeks post-vaccination. The group that was administered the inactivated mutant virus twice exhibited a significantly increased neutralizing antibody titer after a challenge with the virulent homologous strain and exhibited more rapid clearing of viremia compared to other groups, including the groups that were administered either the inactivated mutant or wild-type virus only once and the group that was administered the inactivated wild-type virus twice. Histopathological examination of lung tissue sections revealed that the group that was administered the inactivated mutant virus twice exhibited significantly thinner alveolar septa, whereas the thickness of the alveolar septa of the other groups were markedly increased due to lymphocyte infiltration. These results indicated that the deglycosylation of GP5 enhanced the immunogenicity of the inactivated mutant PRRSV and that twice administrations of the inactivated mutant virus conferred better protection against the homologous challenge. These findings suggest that the inactivated PRRSV that expresses a hypo-glycosylated GP5 is a potential inactivated vaccine candidate and a valuable tool for controlling PRRS for the swine industry.     

In silico prediction and ex vivo evaluation of potential T-cell epitopes in glycoproteins 4 and 5 and nucleocapsid protein of genotype-I (European) of porcine reproductive and respiratory syndrome virus

T-cell epitopes of porcine reproductive and respiratory syndrome virus (PRRSV) glycoproteins 4 (GP4), 5 (GP5) and nucleocapsid (N) were predicted using bioinformatics and later tested by IFN-γ ELISPOT in pigs immunized with either a modified live vaccine (MLV) or DNA (open reading frames 4, 5 or 7). For MLV-vaccinated pigs, immunodominant epitopes were found in N but T-epitopes were also found in GP4 and GP5. For DNA-immunized pigs, some peptides were differently recognized. Using a large set of PRRSV sequences it was shown that N contains a conserved epitope and that for GP5, the genotype-I counterparts of previously reported epitopes of genotype-II strains were also immunogenic.     

Genetic diversity and phylogenetic analysis of porcine reproductive and respiratory syndrome virus isolates in East China

Porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to have evolved at a high evolutionary rate and the extensive genetic variation. In this study, 44 PRRSV positive cases obtained from different provinces of China were sequenced and analyzed. Comparative analysis of partial isolates based on nsp2 sequences revealed that highly pathogenic PRRSV were the dominant viruses in China from 2008 to 2010 and some novel strains with an extra deletion of 19 aa. Phylogenetic analysis based on the GP5 genes showed that the PRRSV isolates from 1996 to 2010 had a great variation and the North American genotype was further divided into six subgenotypes. No apparent relationship between the heterogeneity and the geographic origin of isolates was observed. The 44 isolates and 29 representative strains were divided into six subgenotypes. Further analysis of the GP5 protein suggested that these strains of subgenotypes I, II and III exhibited variations in the primary neutralizing epitope and almost all isolates of subgenotypes II and III had more N-linked glycosylation sites. In addition, some mutations which could mirror the viral evolution and adaptation were also observed in this study. All these results might be useful to study the genetic variation and genetic relatedness among PRRSV strains in China.     

In vivo targeting of porcine reproductive and respiratory syndrome virus antigen through porcine DC-SIGN to dendritic cells elicits antigen-specific CD4T cell immunity in pigs

Immunogenicity of protein subunit vaccines may be dramatically improved by targeting them through antibodies specific to c-type lectin receptors (CLRs) of dendritic cells in mice, cattle, and primates. This novel vaccine development approach has not yet been explored in pigs or other species largely due to the lack of key reagents. In this study, we demonstrate that porcine reproductive and respiratory syndrome virus (PRRSV) antigen was targeted efficiently to dendritic cells through antibodies specific to a porcine CLR molecule DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) in pigs. A recombinant PRRSV antigen (shGP45M) was constructed by fusing secretory-competent subunits of GP4, GP5 and M proteins derived from genetically-shuffled strains of PRRSV. In vaccinated pigs, when the PRRSV shGP45M antigen was delivered through a recombinant mouse-porcine chimeric antibody specific to the porcine DC-SIGN (pDC-SIGN) neck domain, porcine dendritic cells rapidly internalized them in vitro and induced higher numbers of antigen-specific interferon-γ producing CD4T cells compared to the pigs receiving non-targeted PRRSV shGP45M antigen. The pDC-SIGN targeting of recombinant antigen subunits may serve as an alternative or complementary strategy to existing vaccines to improve protective immunity against PRRSV by inducing efficient T cell responses.     

Immune response of pigs inoculated with Mycobacterium bovis BCG expressing a truncated form of GP5 and M protein of porcine reproductive and respiratory syndrome virus

Pigs were immunised with recombinant BCG (rBCG) expressing a truncated form of GP5 (lacking the first 30 NH(2)-terminal residues) (rBCGGP5) and M protein (rBCGM) of porcine reproductive and respiratory syndrome virus (PRRSV). At 30 days post-inoculation (dpi), pigs inoculated with rBCGGP5 and rBCGM developed a specific humoral immune response against the viral proteins, as detected by commercial ELISA and Western blot tests, and at 60 dpi, three out of five animals developed neutralizing antibodies with titers ranging from 1:4 to 1:8. At 67 dpi, an IFN-gamma response against BCG antigens, but not against the viral proteins, was detected by ELISPOT in inoculated pigs. Following challenge with a pathogenic strain of PRRSV, pigs inoculated with rBCG showed lower (P<0.05) temperature, viremia and virus load in bronchial lymph nodes than control animals, suggesting the establishment of partial protection against PRRSV infection.     

Characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies

The porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes reproductive failure in sows and boars, and respiratory disease in pigs of all ages. Antibodies against several viral envelope proteins are produced upon infection, and the glycoproteins GP4 and GP5 are known targets for virus neutralization. Still, substantial evidence points to the presence of more, yet unidentified neutralizing antibody targets in the PRRSV envelope proteins. The current study aimed to identify and characterize linear antigenic regions (ARs) within the entire set of envelope proteins of the European prototype PRRSV strain Lelystad virus (LV). Seventeen LV-specific antisera were tested in pepscan analysis on GP2, E, GP3, GP4, GP5 and M, resulting in the identification of twenty-one ARs that are capable of inducing antibodies upon infection in pigs. A considerable number of these ARs correspond to previously described epitopes in different European- and North-American-type PRRSV strains. Remarkably, the largest number of ARs was found in GP3, and two ARs in the GP3 ectodomain consistently induced antibodies in a majority of infected pigs. In contrast, all remaining ARs, except for a highly immunogenic epitope in GP4, were only recognized by one or a few infected animals. Sensitivity to antibody-mediated neutralization was tested for a selected number of ARs by in vitro virus-neutralization tests on alveolar macrophages with peptide-purified antibodies. In addition to the known neutralizing epitope in GP4, two ARs in GP2 and one in GP3 turned out to be targets for virus-neutralizing antibodies. No virus-neutralizing antibody targets were found in E, GP5 or M. Since the neutralizing AR in GP3 induced antibodies in a majority of infected pigs, the immunogenicity of this AR was studied more extensively, and it was demonstrated that the corresponding region in GP3 of virus strains other than LV also induces virus-neutralizing antibodies. This study provides new insights into PRRSV antigenicity, and contributes to the knowledge on protective immunity and immune evasion strategies of the virus.     

Identification of immunodominant T-cell epitopes present in glycoprotein 5 of the North American genotype of porcine reproductive and respiratory syndrome virus

Ninty-six pentadecapeptides spanning glycoprotein 5 (GP5) of porcine reproductive and respiratory virus (PRRSV) were screened for their ability to elicit a recall interferon-gamma response from peripheral blood mononuclear cells isolated from 22 pigs infected with up to two genetically divergent PRRSV strains. Two distinct regions (amino acid residues 117-131, LAALICFVIRLAKNC, and 149-163, KGRLYRWRSPVII/VEK) appeared to contain immunodominant T-cell epitopes based on their ability to stimulate above average numbers of interferon-gamma secreting cells as compared to other GP5 peptides. A survey of PRRSV isolates indicated that these two sites are relatively conserved with at most a two amino acid variation and thus should be considered for incorporation into a multi-valent vaccine against PRRS.     

A novel baculovirus vector shows efficient gene delivery of modified porcine reproductive and respiratory syndrome virus antigens and elicits specific immune response

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating epizootic of porcine species. Current vaccines are inadequate to control the disease burden and outbreaks in the field. We report a novel baculovirus vaccine vector with White spot syndrome virus immediate early 1 shuttle promoter, with strong activity in both insect cells and mammalian cells, for immunization against PRRSV. The insect cell cultured baculovirus vector produces PRRSV envelope glycoproteins ORF2a, ORF3, ORF4 and ORF5, which are similar to the antigens in the infectious PRRS virion, and these antigens are stably incorporated on the surface of the baculovirus. Further, the baculovirus vector efficiently transduces these antigens in cells of porcine origin, thereby simulating a live infection. The baculovirus vectored PRRSV antigens, upon inoculation in mice, elicits robust neutralizing antibodies against the infective PRRS virus. Further, the experiments indicate that hitherto under emphasized ORF2a and ORF4 are important target antigens for neutralizing PRRSV infectivity.     

Construction and immunogenicity of recombinant Mycobacterium bovis BCG expressing GP5 and M protein of porcine reproductive respiratory syndrome virus

Mycobacterium bovis BCG was used to express a truncated form of GP5 (lacking the first 30 NH(2)-terminal residues) and M protein of porcine reproductive and respiratory syndrome virus (PRRSV). The PRRSV proteins were expressed in BCG under control of the mycobacterial hsp60 gene promoter either in the mycobacterial cytoplasm (BCGGP5cyt and BCGMcyt) or as MT19-fusion proteins on the mycobacterial surface (BCGGP5surf and BCGMsurf). Mice inoculated with BCGGP5surf and BCGMsurf developed antibodies against the viral proteins at 30 days post-inoculation (dpi) as detected by ELISA and Western blot. By 60 dpi, the animals developed titer of neutralizing antibodies of 8. A PRRSV-specific gamma interferon response was also detected in splenocytes of recombinant BCG-inoculated mice at 60 and 90 dpi. These results indicate that BCG was able to express antigens of PRRSV and elicit an immune response against the viral proteins in mice.     

Immunogenicity and protective efficacy of recombinant pseudorabies virus expressing the two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) infection still remains today as the most significant health threat to swine and poses a challenge to current vaccination strategies. To develop a new generation of vaccine against PRRSV, a live attenuated pseudorabies virus (PRV) was used as vaccine vector to express the two major membrane-associated proteins (GP5 or M) of PRRSV in various forms. Four PRV recombinants, rPRV-GP5 (expressing native GP5), rPRV-GP5m (expressing GP5m, a modified GP5), rPRV-GP5-M (co-expressing GP5 and M proteins), rPRV-GP5m-M (co-expressing GP5m and M proteins) were generated. Mouse immunized with all these recombinants developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. However, the highest level of PRRSV-specific neutralizing antibodies and lymphocyte proliferative responses was observed in mice immunized with rPRV-GP5m-M. The immunogenicity and protective efficiency of rPRV-GP5m-M were further evaluated in the piglets. Compared to commercial PRRSV killed vaccine, detectable PRRSV-specific neutralizing antibody and higher lymphocyte proliferative responses could be developed in piglets immunized with rPRV-GP5m-M before virus challenge. Furthermore, more efficient protection against a PRRSV challenge was obtained in piglets immunized with rPRV-GP5m-M, as showed by the balanced body-temperature fluctuation, shorter-term viremia, lower proportion of virus load in nasal and oropharyngeal scrapings and tissues, and milder lung lesions. These data indicate that the recombinant rPRV-GP5m-M is a promising candidate bivalent vaccine against both PRV and PRRSV infection.     

Porcine Reproductive and Respiratory Syndrome Virus isolates differ in their susceptibility to neutralization

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is highly heterogenic. This heterogeneity has an effect on antigenic composition of PRRSV and might create differences in sensitivity to neutralization between isolates. The sensitivity to neutralization could be an important feature of PRRSV isolates because it is likely that isolates resistant to neutralization pose a significant challenge for the development of vaccines that elicit broad protective immunity. Nonetheless, little information is available for understanding or categorizing the viral neutralization phenotype of PRRSV isolates. Consequently, the main purpose of this study was to determine whether PRRSV isolates differ in their susceptibility to neutralization and if they can be classified in different categories based on their neutralization phenotype. For this purpose, a panel of 39 PRRSV isolates and a set of 30 hyperimmune monospecific sera were used in cross-neutralization assays. The results of this study indicate that PRRSV isolates differ in their sensitivity to neutralization and k-means clustering system allowed classifying the isolates in four different categories according to their neutralization phenotype: highly sensitive, sensitive, moderately sensitive and resistant to neutralization. Further analyses using two additional clustering systems that considered individual data for the classification of the isolates confirmed that classification obtained by k-means is accurate in most cases and that only in a few instances classification is less stringent. Sequences of GP3, GP4 and GP5 were analyzed but no correlation could be found between the sequence of previously identified neutralizing epitopes or the number of N-linked glycosylation sites in different proteins and the neutralization phenotype of the isolates. These data provide the first systematic assessment of overall neutralization sensitivities of a panel of diverse PRRSV isolates. The classification of the isolates provides a useful tool to facilitate the systematic characterization of neutralizing antibody production elicited by new vaccine candidates.