Replacement of Porcine CD163 Scavenger Receptor Cysteine-Rich Domain 5 with a CD163-Like Homolog Confers Resistance of Pigs to Genotype 1 but Not Genotype 2 Porcine Reproductive and Respiratory Syndrome Virus.

CD163 knockout (KO) pigs are resistant to infection with genotype 2 (type 2) porcine reproductive and respiratory syndrome virus (PRRSV). Furthermore, the substitution of CD163 scavenger receptor cysteine-rich (SRCR) domain 5 with a homolog of human CD163-like (hCD163L1) SRCR 8 domain confers resistance of transfected HEK cells to type 1 PRRSV. As a means to understand the role of domain 5 in PRRSV infection with both type 1 and type 2 viruses, pigs were genetically modified (GM) to possess one of the following genotypes: complete knockout (KO) of CD163, deletions within SRCR domain 5, or replacement (domain swap) of SRCR domain 5 with a synthesized exon encoding a homolog of hCD163L1 SRCR domain 8. Immunophenotyping of porcine alveolar macrophages (PAMs) showed that pigs with the KO or SRCR domain 5 deletion did not express CD163. When placed in culture, PAMs from pigs with the CD163 KO phenotype were completely resistant to a panel consisting of six type 1 and nine type 2 isolates. PAMs from pigs that possessed the hCD163L1 domain 8 homolog expressed CD163 and supported the replication of all type 2 isolates, but no type 1 viruses. Infection of CD163-modified pigs with representative type 1 and type 2 viruses confirmed the in vitro results. The results confirm that CD163 is the likely receptor for all PRRS viruses. Even though type 1 and type 2 viruses are considered phenotypically similar at several levels, there is a distinct difference between the viral genotypes in the recognition of CD163. IMPORTANCE: Genetic modification of the CD163 gene creates the opportunity to develop production animals that are resistant to PRRS, the costliest viral disease to ever face the swine industry. The results create further opportunities to develop refinements in the modification of CD163 with the goal of making pigs refractory to infection while retaining important CD163 functions.

A single amino acid deletion in the matrix protein of porcine reproductive and respiratory syndrome virus confers resistance to a polyclonal swine antibody with broadly neutralizing activity.

Assessment of virus neutralization (VN) activity in 176 pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) identified one pig with broadly neutralizing activity. A Tyr-10 deletion in the matrix protein provided escape from broad neutralization without affecting homologous neutralizing activity. The role of the Tyr-10 deletion was confirmed through an infectious clone with a Tyr-10 deletion. The results demonstrate differences in the properties and specificities of VN responses elicited during PRRSV infection.

The development of a dexamethasone challenge model for evaluating feed additives in sheep.

OBJECTIVE: To develop an accessible ruminant immune challenge model for rapid in vivo assessments of feed additives. ANIMALS: 60 hair-breed ram lambs. METHODS: Sheep were randomly assigned to 1 of 4 treatments: treatment 1, not immunosuppressed, control fed (n = 12); treatment 2, immunosuppressed, supplemented with a yeast and botanical extract (n = 18); treatment 3, immunosuppressed, supplemented with a blend of natural aluminosilicates and yeast components (n = 18); and treatment 4, immunosuppressed, control fed (n = 12). Twice-daily injections of dexamethasone (Dex; 0.1 mg/kg bodyweight, SC) were used to induce immunosuppression throughout the study (from September 25, 2020, to November 2, 2020). All sheep were immunized with keyhole limpet hemocyanin (KLH) on days 0 and 14 and injected with heat-aggregated KLH, ID, to induce a skin induration on day 15. Measurements included body weight (BW), average daily gain (ADG), CBC, and skin induration diameter. RESULTS: Dex treatment resulted in reduced BW and ADG that was not mitigated by either feed additive. Dex reduced lymphocyte percentage, RBC count, hemoglobin, hematocrit, and skin induration diameter and increased concentrations of granulocytes and granulocyte percentage. Effects on hematocrit, hemoglobin, RBC, and skin induration diameter were mitigated with the addition of feed additives. CLINICAL RELEVANCE: The described model is a tool to evaluate the ability of feed additives to mitigate the immunosuppressive effects of Dex.

Recognition of the different structural forms of the capsid protein determines the outcome following infection with porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) capsid protein (CP) is the only protein necessary for the formation of the virion capsid, and recombinant CP spontaneously forms virus-like particles (VLPs). Located within a single CP subunit is an immunodominant epitope consisting of residues 169 to 180 [CP(169-180)], which is exposed on the surface of the subunit, but, in the structural context of the VLP, the epitope is buried and inaccessible to antibody. High levels of anti-CP(169-180) activity are associated with porcine circovirus-associated disease (PCVAD). The purpose of this study was to investigate the role of the immune response to monomer CP in the development of PCVAD. The approach was to immunize pigs with CP monomer, followed by challenge with PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV). To maintain the CP immunogen as a stable monomer, CP(43-233) was fused to ubiquitin (Ub-CP). Size exclusion chromatography showed that Ub-CP was present as a single 33-kDa protein. Pigs immunized with Ub-CP developed a strong antibody response to PCV2, including antibodies against CP(169-180). However, only low levels of virus neutralizing activity were detected, and viremia levels were similar to those of nonimmunized pigs. As a positive control, immunization with baculovirus-expressed CP (Bac-CP) resulted in high levels of virus neutralizing activity, small amounts of anti-CP(169-180) activity, and the absence of viremia in pigs following virus challenge. The data support the role of CP(169-180) as an immunological decoy and illustrate the importance of the structural form of the CP immunogen in determining the outcome following infection.

Identification and characterization of the causative agents of Focal Ulcerative Dermatitis in commercial laying hens.

Focal Ulcerative Dermatitis (FUDS) is an emerging dermatological disease that affects cage-free laying flocks, it is characterized by the development of a lesion on the dorsum of the birds; FUDS is sporadic in nature and can result in a drop in egg production and up to 50% of cumulative mortality. A total of two cage-free flocks (flock 1: no history of FUDS; flock 2: birds affected with FUDS) from a commercial laying hen operation in the mid-west U.S. were sampled in this study. The microbial composition of skin, cloacal, cecal, and ileal samples from each bird was characterized through next generation sequencing (NGS). Results identified Staphylococcus aureus and Staphylococcus agnetis as the potential causative agents of FUDS, being the most predominant in FUDS positive birds. These results were confirmed by plating, with both staphylococci as the only pathogens isolated from lesions of FUDS positive birds. A total of 68 confirmed Staphylococcus isolates from skin and environmental samples were further analyzed by whole genome sequencing (WGS) for the presence of antimicrobial resistance (AMR) genes and virulence factors that could have contributed to the development of FUDS. Forty-four-point one-two percent of the isolates had between one and four acquired AMR genes encoding for macrolides, lincosamides, spectrogramines, and beta-lactams resistance. Six classes of virulence factors associated with adherence, enzyme, immune evasion, secretion system, toxin, and iron uptake were identified. The antimicrobial effect of 4 proprietary Bacillus Direct Fed Microbial (DFM) combinations was evaluated against the Staphylococcus aureus and Staphylococcus agnetis isolates, by agar well-diffusion (AWD) assay and competitive exclusion (CE) on broth culture. Through this antimicrobial screening, a particular two-strain combination of Bacillus pumilus was identified as the most effective inhibitor of both staphylococci. A customized Bacillus pumilus product is being used at different farms with history of FUDS resulting in the successful inhibition of both Staphylococcus aureus and Staphylococcus agnetis, decreasing FUDS mortalities, and improving harvestable eggs.

Amplification and selection of PRRSV-activated VDJ repertoires in pigs secreting distinct neutralizing antiboidies.

Neutralizing antibodies (nAbs) play an important role in protective immunity against porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, the characterization of PRRSV nAb repertoires is rarely investigated. In this study, we developed a swine VDJ amplification method and selection criteria for the characterization of PRRSV-activated VDJ repertoires. According to clonal expansion theory, two separated aliquots of lymph nodes from pigs producing different PRRSV nAbs were utilized to determine the activated B-cell repertoires. Swine VDJ repertoires from a mock-infected pig and PRRSV-infected pigs secreting no detectable nAbs, only homologous nAbs, and broad nAbs were amplified by a single pair of primers that could detect all seven major VDJ genes. The amplicons were cloned and sequenced to generate 385 VDJ sequences. Sequence alignment showed that the diversification of VDJ genes was mainly due to the variation in complementarity determining regions (CDRs), especially CDR3. Based on selection criteria, shared and abundant sequences were identified in two separated aliquots from PRRSV-infected pigs but not from the mock-infected pig, suggesting they were secreted from PRRSV-activated B cells. Thus, the amplification and selection method provide a potential alternative for the characterization of swine VDJ repertoires. However, additional experiments are required to determine whether the shared and abundant VDJ lineages identified in this study are PRRSV-specific or distinct neutralizing-antibodies-associated.

GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) as a target for homologous and broadly neutralizing antibodies.

Virus neutralization (VN) responses range from narrowly focused antibodies with only homologous neutralizing activity against the virus used for infection, to antibodies that can neutralize both Type 1 and Type 2 viruses, referred to as broadly neutralizing antibody (bnAb). Even though neutralizing epitopes are likely distributed among several structural glycoproteins, this paper focuses on the ectodomain region of GP5 as a model system for investigating the role for neutralizing and non-neutralizing antibodies in protection and disease. Epitope B within GP5 possesses several features common to broadly neutralizing epitopes. In the proposed model, accessibility of antibody to Epitope B is blocked by homologous neutralizing and non-neutralizing antibodies, which bind flanking hypervariable domains. Additional mechanisms for blocking the accessibility of bnAb include conformational alterations within the GP5-M heterodimer and glycan shielding. This model explains how the continuous escape from homologous neutralization provides a mechanism for persistence. The proposed mechanism for immune evasion is not unique to PRRSV, but can be found in other persistent viruses, such as hepatitis C virus (HCV).

ORF5 of porcine reproductive and respiratory syndrome virus (PRRSV) is a target of diversifying selection as infection progresses from acute infection to virus rebound.

Genetic variation in both structural and nonstructural genes is a key factor in the capacity of porcine reproductive and respiratory syndrome virus (PRRSV) to evade host defenses and maintain within animals, farms and metapopulations. However, the exact mechanisms by which genetic variation contribute to immune evasion remain unclear. In a study to understand the role of host genetics in disease resistance, a population of pigs were experimentally infected with a type 2 PRRSV isolate. Four pigs that showed virus rebound at 42days post-infection (dpi) were analyzed by 454 sequencing to characterize the rebound quasispecies. Deep sequencing of variable regions in nsp1, nsp2, ORF3 and ORF5 showed the largest number of nucleotide substitutions at day 28 compared to days 4 and 42 post-infection. Differences were also found in genetic variations when comparing tonsil versus serum. The results of dN/dS ratios showed that the same regions evolved under negative selection. However, eight amino acid sites were identified as possessing significant levels of positive selection, including A27V and N32S substitutions in the GP5 ectodomain region. These changes may alter GP5 peptide signal sequence processing and N-glycosylation, respectively. The results indicate that the greatest genetic diversity occurs during the transition between acute and rebound stages of infection, and the introduction of mutations that may result in a gain of fitness provides a potential mechanism for persistence.

Vaccination with a Porcine Reproductive and Respiratory Syndrome (PRRS) Modified Live Virus Vaccine Followed by Challenge with PRRS Virus and Porcine Circovirus Type 2 (PCV2) Protects against PRRS but Enhances PCV2 Replication and Pathogenesis Compared to Results for Nonvaccinated Cochallenged Controls.

Coinfections involving porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) contribute to a group of disease syndromes known as porcine circovirus-associated disease (PCVAD). Presumably, PRRSV infection enhances PCV2 replication as a result of modulation of host immunity. The purpose of this study was to evaluate PCV2 replication and pathogenesis in pigs vaccinated with a PRRS modified live virus (MLV) vaccine and subsequently challenged with a combination of PRRSV and PCV2. During the early postchallenge period, the number of pigs with PRRSV-associated clinical signs was decreased, and average daily gain (ADG) was increased, in the vaccinated group, demonstrating the protective effect of PRRS vaccination. However, during the later postchallenge period, more pigs in the vaccinated group showed increased PCV2 viremia, decreased ADG, increased PCVAD clinical signs, and increased mortality. In this disease model, the early benefits of PRRSV vaccination were outweighed by the later amplification of PCVAD.

Multiplex serology for common viral infections in feral pigs (Sus scrofa) in Hawaii between 2007 and 2010.

Multiplex serology was performed for the detection of total immunoglobulin (Ig) and IgM antibodies against porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and swine influenza virus (SIV) antigens in feral swine (Sus scrofa). Serum samples were collected from the islands of Oahu (292 pigs) and Hawaii (52 pigs) between 2007 and 2010. The highest antibody prevalence was to PCV2 (63%), followed by SIV (7.8%) and PRRSV (5.8%). Antigen-specific IgM was detected at a much lower prevalence. PCR amplification and sequence analysis of PCV2 in three IgM-positive samples identified PCV2b as the only genotype. While the prevalence of PCV2 and PRRSV remained similar between 2007 and 2010, the percentage of SIV-positive samples on Oahu increased from 2% to 19%. Our results demonstrate the utility of multiplex serology for pathogen surveillance in feral pig populations.

Genetic variation of porcine circovirus type 2 (PCV2) and its relevance to vaccination, pathogenesis and diagnosis.

Porcine circovirus-associated disease (PCVAD) encompasses a group of complex, multi-factorial syndromes, which are dependent on infection with porcine circovirus type 2 (PCV2). Current strains of PCV2 circulating in the field are classified into two groups, termed PCV2a and PCV2b. Outbreaks of severe PCVAD in North America and other countries are often linked to a shift from PCV2a to PCV2b as the predominant genotype. Therefore, genotype-specific differences in pathogenesis and antigenicity have been suggested. Overall, evidence suggests that virulence is a function of the specific PCV2 isolate, regardless of genotype. In addition, only minor antigenic differences have been reported. In terms of immunopathogenesis, a conserved decoy epitope, located in the C-terminal region of the capsid protein, provides an explanation for the inability to identify pathogenic differences between genotypes. Finally, genetic variation in PCV2 and the resulting consequences with respect to vaccination and diagnostics are discussed.

Pathogenesis of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most costly viral pathogen facing a modern pig industry. A unique feature of the virus is the ability to cause severe clinical disease and maintain a life-long subclinical infection. Persistence at the population level poses the biggest challenge for the successful control and elimination of the disease. A mechanistic basis for persistence includes the evasion of innate and adaptive immune responses. Recent advances include the study of how the non-structural proteins (nsp's) inhibit the induction of type 1 interferon genes.

Antibody responses following vaccination versus infection in a porcine circovirus-type 2 (PCV2) disease model show distinct differences in virus neutralization and epitope recognition.

Porcine circovirus associated disease (PCVAD) encompasses a group of syndromes linked to infection with porcine circovirus type 2 (PCV2). Based on the hypothesis that the immune responses to vaccination versus infection are quantitatively and qualitatively different, the objective of this study was to evaluate immunity, virus replication and disease protection in pigs vaccinated with PCV2 capsid protein (CP) and during infection. The disease model included dual infection with PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV), a virus known to enhance disease progression and severity. The principal effect of PRRSV infection was to increase peak PCV2 viremia by almost 40-fold; however, PCV2 failed to show a reciprocal effect on PRRSV. In vaccinated pigs, there was no evidence of disease or PCV2 replication following dual virus challenge. Immunity following vaccination favored PCV2 neutralizing activity; whereas, PCV2 infection and disease produced high levels of non-neutralizing antibody, primarily directed against a polypeptide in the C-terminal region of CP. These results support the notion that the magnitude of the total antibody response cannot be used as a measure of protective immunity. Furthermore, protection versus disease lies in the immunodominance of specific epitopes. Epitope specificity should be taken into consideration when designing PCV2 vaccines.

Antibody recognition of porcine circovirus type 2 capsid protein epitopes after vaccination, infection, and disease.

Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233-amino-acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs and to compare these responses to those of pigs diagnosed with porcine circovirus-associated disease (PCVAD), including porcine multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). The approach was to react porcine sera with CP polypeptide fragments followed by finer mapping studies using overlapping oligopeptides that covered amino acids 141 to 200. The results showed that vaccinated pigs preferentially recognized only the largest polypeptide fragment, CP(43-233). A subset of experimentally infected pigs and pigs with PDNS showed strong reactivity against a CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175, and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The recognition of CP(169-180) and other polypeptides provides opportunities to devise diagnostic tests for monitoring the immunological effectiveness of vaccination.