Evaluating an automated clustering approach in a perspective of ongoing surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) field strains

Porcine reproductive and respiratory syndrome virus (PRRSV) has a major economic impact on the swine industry. The important genetic diversity needs to be considered for disease management. In this regard, information on the circulating endemic strains and their dispersal patterns through ongoing surveillance is beneficial. The objective of this project was to classify Quebec PRRSV ORF5 sequences in genetic clusters and evaluate stability of clustering results over a three-year period using an in-house automated clustering system. Phylogeny based on maximum likelihood (ML) was first inferred on 3661 sequences collected in 1998–2013 (Run 1). Then, sequences collected between January 2014 and September 2016 were sequentially added into 11 consecutive runs, each one covering a three-month period. For each run, detection of clusters, which were defined as groups of ≥15 sequences having a≥70% rapid bootstrap support (RBS) value, was automated in Python. Cluster stability was described for each cluster and run based on the number of sequences, RBS value, maximum pairwise distance and agreement in sequence assignment to a specific cluster. First and last run identified 29 and 33 clusters, respectively. In the last run, about 77% of the sequences were classified by the system. Most clusters were stable through time, with sequences attributed to one cluster in Run 1 staying in the same cluster for the 11 remaining runs. However, some initial groups were further subdivided into subgroups with time, which is important for monitoring since one specific wild-type cluster increased from 0% in 2007 to 45% of all sequences in 2016. This automated classification system will be integrated into ongoing surveillance activities, to facilitate communication and decision-making for stakeholders of the swine industry.     

ORF5-based evolutionary and epidemiological dynamics of the type 1 porcine reproductive and respiratory syndrome virus circulating in Korea

This study applied a number of advanced genetic analysis tools to investigate the evolutionary trajectories and epidemiological dynamics of Korean type 1 PRRSV based on variations in the ORF5 gene over a long-term period from 2005 to 2013. Maximum likelihood phylogenetic analysis performed on large, worldwide ORF5 sequences (n = 1127) strongly suggested no further introduction of genetically novel type 1 PRRSV into Korean pig farms, with the identification of only two clusters (I and II) in circulation to date. Using a codon-based extension of the Bayesian relaxed clock model, this study was able to distinguish between synonymous and non-synonymous substitutions and demonstrated that, while the absolute rates of synonymous substitution (E[S]) were similar between clusters I and II, the absolute rate of non-synonymous substitution (E[N]) was significantly different between the clusters. Cluster I was found to have an elevated E[N]/E[S] ratio relative to cluster II on the internal branches, compared to the external branches. Additionally, many fewer sites were predicted under diversifying selection in cluster II than in cluster I. Utilizing the Bayesian skyride method and the novel Bayesian birth–death skyline plot method, this study provided insights into the epidemiological dynamics of type 1 PRRSV in Korea by revealing that each cluster experienced a unique epidemic growth and by uncovering correlations between the effective population size and effective reproductive number.     

Recombination in lineage 1, 3, 5 and 8 of porcine reproductive and respiratory syndrome viruses in China

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important viral swine diseases, resulting in immense economic losses in Chinese pig industry. Currently, four major lineages: lineage 1 (NADC30-like), 3 (QYYZ-like), 5.1 (VR2332-like) and 8.7 (JXA1-like) of type 2 PRRSV (North American type) have been circulating in China based on classification system, which have caused concern about the potential of virus recombination. In the present study, a novel variant of PRRSV strain named FJLIUY-2017 was isolated from abortion rate (25%) in pregnant gilts in Fujian Province in China in 2017. To further our knowledge about the novel virus strain, we characterized the complete genome of FJLIUY-2017. Comparison to PRRS sequences in GenBank confirmed the absence of close relatives (<92%), but indicated FJLIUY-2017 belonged to NADC30-like PRRSV. The full length of FJLIUY-2017 was determined to be 15017 nucleotides (nt), excluding the poly(A) tail, shared 86.2–86.6% identity with JXA1-like strains (JXA1, TJ and FJYR), 88.9–90.6% with NADC30-like PRRSVs (NADC30, FJZ03 and CHsx1401), 86.4–86.5% with VR2332-like (VR2332, RespPRRS MLV and BJ-4) and only 60.8% with LV (European type). Recombination analyses revealed genomic breakpoints in structural (ORF3, ORF4 and ORF7) and nonstructural (Nsp1, Nsp2, Nsp6, Nsp9, Nsp11 and Nsp12) regions of the genomes with evidence for recombination events between lineages 1, 3, 5.1 and 8.7. Taken altogether, the results of our study provide further confirmation that PRRSV is prone to undergo recombination events. Thus, it is critical to monitor PRRSV evolution in China and establish an effective strategy for the control of PRRS.     

Efficacy of a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in pigs naturally exposed to a heterologous European (Italian cluster) field strain: Clinical protection and cell-mediated immunity

The purpose of this study was to assess clinical protection in pigs vaccinated with a commercially available attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (Porcilis^® PRRS) and then naturally exposed under field conditions to a heterologous (Italian cluster) strain of virulent PRRSV. A total of 30, 4-week-old pigs seronegative for PRRSV were allocated to 1 of 3 groups (IM, ID, and C groups). At 5 weeks of age, pigs of groups IM (n = 10 pigs) and ID (n = 10 pigs) were vaccinated intramuscularly and intradermally, respectively, with modified live PRRSV-1 vaccine (Porcilis^® PRRS). Pigs of group C (n = 10 pigs) were kept as non-vaccinated controls. At post-vaccination (PV) days 0, 7, 14, 28, and 45, blood samples were collected for detection of vaccine virus (PCR) and antibody response (ELISA), identification of changes in lymphocyte subpopulations by cytometry, and IFN-γ PRRSV-specific secreting cells (SC) by ELISpot. At PV day 45, pigs of A, B, and C groups were moved to a site 3 conventional finishing herd with a history of respiratory disease caused by PRRSV and the most common bacteria to be exposed to a natural challenge. The PRRSV field strain, belonging to the Italian cluster of the PRRSV-1, demonstrated a 84% identity with the vaccine virus (DV strain) at ORF5 sequencing. At 0 (exposure day = 45 days PV), 4, 7, 11, 14, 19, 21, 28, and 34 days post-exposure (PE) blood samples were collected for detection and titration of PRRSV and antibody, as well as for lymphocyte and IFN-γ measurement as described above. Throughout the post-exposure period, all pigs were observed daily for clinical signs. The overall clinical signs were reduced by 68 and 72%, respectively in the intramuscularly and intradermally vaccinated pigs compared to controls. Respiratory signs were reduced by 72 and 80%, respectively in the IM and ID groups. Clinical protection was associated with marked activation of cell-mediated immune response. The highest levels of specific IFN-γ production at 21–34 days PE were concomitant and associated to changes in natural killer (NK) cells, γ/δ T, and cytotoxic T lymphocytes in the blood. In our field study, evidences of EU attenuated vaccine-induced clinical protection against natural exposure to a genetically diverse (84% homology) PRRSV-1 isolate (Italian cluster) was demonstrated by the statistically significant reduction in clinical signs in terms of incidence, duration and severity and by a more efficient cell-mediated immune response in the vaccinated pigs as compared to the unvaccinated controls.     

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.     

Purifying selection in porcine reproductive and respiratory syndrome virus ORF5a protein influences variation in envelope glycoprotein 5 glycosylation

Porcine reproductive and respiratory syndrome virus ORF5a protein is encoded in an alternate open reading frame upstream of the major envelope glycoprotein (GP5) in subgenomic mRNA5. Bioinformatic analysis of 3466 type 2 PRRSV sequences showed that the two proteins have co-evolved through a fine balance of purifying codon usage to maintain a conserved RQ-rich motif in ORF5a protein, while eliciting a variable N-linked glycosylation motif in the alternative GP5 reading frame. Conservation of the ORF5a protein RQ-motif also explains an anomalous uracil desert in GP5 hypervariable glycosylation region. The N-terminus of the mature GP5 protein was confirmed to start with amino acid 32, the hypervariable region of the ectodomain. Since GP5 glycosylation variability is assumed to result from immunological selection against neutralizing antibodies, these findings show that an alternative possibility unrelated to immunological selection not only exists, but provides a foundation for investigating previously unsuspected aspects of PRRSV biology. Understanding functional consequences of subtle nucleotide sequence modifications in the region responsible for critical function in ORF5a protein and GP5 glycosylation is essential for rational design of new vaccines against PRRS.     

Attempts to enhance cross-protection against porcine reproductive and respiratory syndrome viruses using chimeric viruses containing structural genes from two antigenically distinct strains

Due to significant antigenic variations between field isolates of porcine reproductive and respiratory syndrome virus (PRRSV), suboptimal cross-protection between different viruses impedes the effective control of PRRS via vaccination. Our previous study showed that chimeric viruses containing mixed structural genes from two distinct strains (VR2332 and JA142) of PRRSV were highly susceptible to the viral neutralizing activity of antisera generated against both parental strains. In this study, three chimeric viruses (JAP5, JAP56 and JAP2–6) were constructed by replacing ORF5, ORFs 5 and 6, and ORFs 2-6 of VR2332 with the corresponding genes of JA142, respectively, and their ability to confer cross-protection against challenge with the VR2332 and JA142 strains was evaluated in vivo. A total of 114 pigs were divided into 6 groups, and each group was intramuscularly injected with one of the 3 chimeric viruses (n = 16 pigs per group), VR2332 (n = 24), JA142 (n = 24), or sham inoculum (n = 18). At 44 days post-inoculation (dpi), these pigs were further divided into 15 groups (n = 6 or 8 pigs per group) and intranasally challenged with VR2332, JA142, or sham inoculum. All pigs inoculated with one of the chimeric viruses prior to challenge had lower viremia levels than the challenge control pigs. Prior inoculation with JAP56 markedly decreased viremia to nearly undetectable levels in pigs challenged with either VR2332 or JA142. These results suggest that chimeric viruses harboring mixed structural genes from two distinct PRRSV strains can provide protection against both donor viruses.     

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.     

Vaccine efficacy of porcine reproductive and respiratory syndrome virus chimeras

The vaccine efficacy of six PRRSV Type 2 infectious clones, including five chimeras and a strain-specific deletion mutant, were examined using a respiratory challenge model in growing swine. The chimeras were constructed from different combinations of a licensed modified live vaccine (Ingelvac^® PRRS MLV) and a virulent field isolate (wt MN184) which differ by 14.3% on a nucleotide basis, while the deletion mutant tested had a broad deletion in the nsp2 region of strain MN184. The appearance of antibodies and virus characterization revealed regions of the genome that could influence PRRSV replication in vivo. Swine growth, clinical signs and lung lesions were also monitored. Average daily weight gain was negatively and directly impacted by some vaccines, and after challenge, vaccination with different constructs led to variable weight gain. We determined that 3 of the tested chimeras, including two previously published chimeras [1] and one in which strain MN184 ORF5-6 was placed on the background of Ingelvac^® PRRS MLV were able to prevent lung consolidation to a similar extent as traditionally prepared cell-passaged attenuated vaccines. The study suggested that only specific chimeras can attenuate clinical signs in swine and that attenuation cannot be directly linked to primary virus replication. Additionally, the strain MN184 deletion mutant was not found to have been sufficiently attenuated nor efficacious against heterologous challenge with strain JA-142.     

Genetic diversity of the Korean field strains of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically significant diseases in the swine industry. The PRRS virus (PRRSV) has genetically diverse populations, like other RNA viruses, and various field strains continue to be reported worldwide. The molecular epidemiological study of PRRSV can provide important data for use in controlling the disease. In this study, 50 oral fluid samples from conventional farms in Korea were taken to analyze nucleotide sequences of the open reading frame 5 of PRRSV. The viruses present in more than 80% of oral fluid samples genetically originated from the type 2 PRRSV, which is North American (NA) lineage. In addition 8.9% of samples contained both of the type 1 PRRSV, which is European (EU) lineage and the type 2 PRRSV. About 60% of farms involved in this study had more than two strains of PRRSV. In phylogenetic analysis, the Korean field strains of PRRSV detected from the oral fluid samples were divided into several subgroups: four subgroups of Korean field strains clustered with the type 1 PRRSV, and other five subgroups of Korean field strains clustered with the type 2. These results suggest that the type 2 PRRSV is more prevalent than the type 1 in Korea and heterologous strains of PRRSV can simultaneously infect a single pig farm.     

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.     

In vitro and in vivo studies of deglycosylated chimeric porcine reproductive and respiratory syndrome virus as a vaccine candidate and its realistic revenue impact at commercial pig production level

Porcine reproductive and respiratory syndrome virus (PRRSV) causes major economic losses in the swine industry worldwide. Vaccination is the most effective method to control the disease. In a previous study, a chimeric PRRSV named as K418 which had a genome composed of ORF 1 from the FL12 strain and ORF 2-7 from the Korean representative LMY strain was created. We constructed K418DM, K418 with deglycosylated glycoprotein 5 (GP5), to improve its humoral immunity. In the follow-up on in vivo and in vitro virological and serological tests, no back mutation in amino acids of GP5 associated with deglycosylation was shown after 9 passages on MARC-145 cells, whereas only one case of back mutation was detected after single passage in pig. In serological study, K418DM induced higher serum neutralization (SN) antibody and more limited viremia compared with those of K418 virus. In clinical trial and economic analysis, the K418DM elicited SN antibody titers and PRRSV-specific IgG over protection limit. From the economic viewpoint, there was statistically significant reduction in percentage of weak pigs. These results indicated that vaccination with the K418DM may provide enhanced protection for pigs in PRRS endemic situation and increase growth performance in commercial pig farms.     

Protection and immune response in pigs intradermally vaccinated against porcine reproductive and respiratory syndrome (PRRS) and subsequently exposed to a heterologous European (Italian cluster) field strain

The purpose of this study was to assess the immune response in pigs intradermally vaccinated with a commercially available attenuated porcine reproductive and respiratory virus (PRRSV) vaccine (Porcilis PRRS) and subsequently exposed to a heterologous (Italian cluster) field strain of virulent PRRSV. A total of 18, 4-week-old pigs seronegative for PRRSV were allocated to 1 of 3 groups (groups A, B, and C). At 5 weeks of age, pigs of groups A (n=6 pigs) and B (n=6 pigs) were vaccinated intramuscularly and intradermally, respectively, with Porcilis PRRS. The more conventional intramuscular route of vaccination was included for comparative purposes with the intradermal route of vaccination (performed with the I.D.A.L. vaccinator). Pigs of group C (n=6 pigs) were kept as nonvaccinated controls. At post-vaccination (PV) days 7, 14, 21, 28, and 35, blood samples were collected for detection of vaccine virus (PCR) and antibodies (ELISA), and for changes in PBMC (flow cytometry). At PV day 35, pigs of all groups were each exposed (challenged) intranasally to a heterologous field strain (78% ORF5 sequence homology between vaccine and field virus) belonging to the Italian cluster of the European genotype of PRRSV. At post-challenge (PC) days 0, 3, 7, 10, 13, and 17, blood samples were collected for detection and quantitation of virus and antibodies, and for changes in PBMC as described above for blood samples collected PV. Throughout the experiment all pigs were observed daily for clinical signs. At PC days 7 and 17, two pigs and four pigs, respectively, of each group were euthanized and examined for macroscopic lesions. Following vaccination some pigs of groups A and B had a detectable viremia that in two pigs (one pig of group A and one pig of group B) lasted until PV day 28. However, all pigs (groups A, B, and C) remained clinically normal. All vaccinated pigs developed a serological response (ELISA) to PRRSV. Presumptive evidence for vaccine-induced protective immunity against the heterologous challenge strain was provided by finding that viremia following challenge was generally less (incidence) and significantly less (titers) in vaccinated pigs than in nonvaccinated pigs. No differences were apparent between pigs vaccinated intramuscularly and those vaccinated intradermally. The absence of virulent-virus-induced clinical signs and macroscopic lesions in nonvaccinated as well as in vaccinated pigs precluded a more definitive evaluation of the magnitude of protective immunity provided by vaccination or by the route of vaccination. Some likely treatment-associated changes in lymphocyte subpopulations were observed among the three treatment groups. These changes and their potential relationship to protective immunity are discussed.     

Cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive chronic respiratory viral disease of pigs that is responsible for major economic losses to the swine industry worldwide. The efficacy of parenteral administration of widely used modified live virus PRRS vaccine (PRRS-MLV) against genetically divergent PRRSV strains remains questionable. Therefore, we evaluated an alternate and proven mucosal immunization approach by intranasal delivery of PRRS-MLV (strain VR2332) with a potent adjuvant to elicit cross-protective immunity against a heterologous PRRSV (strain MN184). Mycobacterium tuberculosis whole cell lysate (Mtb WCL) was chosen as a potent mucosal adjuvant due to its Th1 biased immune response to PRRS-MLV. Unvaccinated pigs challenged with MN184 had clinical PRRS with severe lung pathology; however, vaccinated (PRRS-MLV+ Mtb WCL) pigs challenged with MN184 were apparently healthy. There was a significant increase in the body weight gain in vaccinated compared to unvaccinated PRRSV challenged pigs. Vaccinated compared to unvaccinated, virus-challenged pigs had reduced lung pathology associated with enhanced PRRSV neutralizing antibody titers and reduced viremia. Immunologically, an increased frequency of Th cells, Th/memory cells, γδ T cells, dendritic cells, and activated Th cells and a reduced frequency of T-regulatory cells were detected at both mucosal and systemic sites. Further, reduced secretion of immunosuppressive cytokines (IL-10 and TGF-β) and upregulation of the Th1 cytokine IFN-γ in blood and lungs were detected in mucosally vaccinated, PRRSV-challenged pigs. In conclusion, intranasal immunization of pigs with PRRS-MLV administered with Mtb WCL generated effective cross-protective immunity against PRRSV.     

Impact of genetic diversity of European-type porcine reproductive and respiratory syndrome virus strains on vaccine efficacy

The aim of this study was to find out how efficiently pigs that are vaccinated with an attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine based on a virus from the Lelystad cluster are protected against a European wild-type strain from the same or another genetic cluster. Two experiments were performed. In each experiment, 5-week-old PRRSV-seronegative pigs were vaccinated intramuscularly with 10(4.5) TCID50 of a commercial vaccine based on a European virus strain from the Lelystad cluster. Non-vaccinated pigs were included as controls. At 5, 9, 15, 20, 28, 35 and 42 days post vaccination (PV), broncho-alveolar lavage (BAL) fluids and blood were collected to determine vaccine virus quantities. Forty-nine days PV, pigs were challenged intranasally with 10(6.0) TCID50 of a European wild-type strain, belonging either to the Lelystad cluster (98% nucleotide identity in ORF5 with vaccine strain) (experiment A) or to an Italian cluster (84% nucleotide identity in ORF5 with vaccine strain) (experiment B). At 5, 9, 15, 20 and 27 days post challenge (PC), BAL fluids and blood were collected to determine virus quantities. Vaccine virus was first detected in BAL fluids and blood at 5 days PV and reached highest quantities between 9 and 15 days PV. One pig was positive in its BAL fluid until 42 days PV. After challenge, virus was isolated from BAL fluids and blood of all non-vaccinated control pigs. All vaccinated pigs challenged with the Lelystad strain remained negative for virus, while virus was present in BAL fluids and blood of all vaccinated pigs after challenge with the Italian strain. Mean virus titres of the vaccinated pigs challenged with the Italian strain were significantly lower than those of the non-vaccinated control pigs (P <0.05) at 9, 15 and 20 days PC. Thus, the genetic diversity within European-type PRRSV may affect the efficacy of the current European-type vaccines.     

Intranasal delivery of whole cell lysate of Mycobacterium tuberculosis induces protective immune responses to a modified live porcine reproductive and respiratory syndrome virus vaccine in pigs

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease to pork producers worldwide. Commercially, both live and killed PRRSV vaccines are available to control PRRS, but they are not always successful. Based on the results of mucosal immunization studies in other viral models, a good mucosal vaccine may be an effective way to elicit protective immunity to control PRRS outbreaks. In the present study, mucosal adjuvanticity of Mycobacterium tuberculosis whole cell lysate (Mtb WCL) was evaluated in pigs administered a modified live PRRS virus vaccine (PRRS-MLV) intranasally. A Mtb WCL mediated increase in the frequency of NK cells, CD8⁺and CD4⁺ T cells, and γδ T cells in pig lungs were detected. Importantly, an increased and early generation of PRRSV specific neutralizing antibodies were detected in PRRS-MLV+ Mtb WCL compared to pigs inoculated with vaccine alone. In addition, there was an increased secretion of Th1 cytokines (IFNγ and IL-12) that correlated with a reciprocal reduction in the production of immunosuppressive cytokines (IL-10 and TGFβ) as well as T-regulatory cells in pigs vaccinated with PRRS-MLV+ Mtb WCL. Further, a complete rescue in arginase levels in the lungs mediated through Mtb WCL was observed in pigs inoculated with PRRS-MLV. In conclusion, Mtb WCL may be a potent mucosal adjuvant for PRRS-MLV in order to potentiate the anti-PRRSV specific immune responses to control PRRS effectively.     

Novel Nsp2 deletion based on molecular epidemiology and evolution of porcine reproductive and respiratory syndrome virus in Shandong Province from 2013 to 2014

Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease affecting swine worldwide. In this study, a total of 385 samples were collected from Shandong pig farms during 2013 and 2014, when pigs were not inoculated with any vaccine. Results indicated that, out of 385 samples, 47 (12.21%) were PRRSV-RNA-positive. The gene sequence analysis of 12 ORF5, 12 ORF7, and 8 Nsp2 of these samples was used to determine the molecular epidemiology of PRRSV in different parts of China’s Shandong Province. The phylogenetic tree based on these 3 genes indicated that the Chinese PRRSV strains could be divided into five subgroups and two large groups. The 8 study strains were clustered into subgroup IV, another 4 strains into subgroup I. The first 8 strains shared considerable homology with VR-2332 in ORF5 (96–97.5%), the other 4 strains shared considerable homology with JXA1 (94–98%). Phylogenetic tree of GP5 showed that the eight isolates formed a tightly novel clustered branch, subgroup V, which resembled but differed from isolate VR-2332. When examined using Nsp2 alone, the first 8 strains showed considerable homology with a U.S. vaccine strain, Ingelvac MLV (89.6–98.4%). One novel pattern of deletion was observed in Nsp2. The genetic diversity of genotype 2 PRRSV tended to vary in the field. The emergence of novel variants will probably be the next significant branch of PRRSV study.     

Live porcine reproductive and respiratory syndrome virus vaccines: Current status and future direction

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.     

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.