Type I interferon suppression-negative and host mRNA nuclear retention-negative mutation in nsp1ß confers attenuation of porcine reproductive and respiratory syndrome virus in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) has the ability to suppress the type I interferons (IFNs-α/ß) induction to facilitate its survival during infection, and the nsp1 protein of PRRSV has been identified as the potent IFN antagonist. The nsp1ß subunit of nsp1 has also been shown to block the host mRNA nuclear export as one of the mechanisms to suppress host antiviral protein production. The SAP motif in nsp1ß is the functional motif for both IFN suppression and host mRNA nuclear retention, and using infectious clones, two mutant viruses vL126A and vL135A have been generated. These mutants retain the infectivity, but the phenotype is negative for both IFN suppression and host mRNA nuclear retention due to the loss of the SAP motif. To examine the pathogenic role of IFN suppression in pigs, 40 piglets were allotted to four groups and each group was intramuscularly infected with vL126A, vL135A, wild-type (WT) PRRSV, and placebo. Pigs infected with vL126A or vL135A exhibited mild clinical signs with low viral titers and short duration of viremia. The levels of PRRSV-specific antibody remained comparable in all infected groups but the neutralizing antibody titers were high in vL126A-infected or vL135A-infected pigs. The IFN-α concentration was also high in pigs infected with the SAP mutants. Reversion to WT sequence was observed in the SAP motif in some animals, and the revertants regained the function to suppress IFN production and host mRNA nuclear export, indicating strong selection pressure in the SAP motif of nsp1ß. Together, our data demonstrate that the IFN antagonism and host mRNA nuclear retention mediated by nsp1ß contributes to viral virulence, and loss of these functions confers PRRSV attenuation.

Genomic regions associated with host response to porcine reproductive and respiratory syndrome vaccination and co-infection in nursery pigs.

BACKGROUND: The WUR1000125 (WUR) single nucleotide polymorphism (SNP) can be used as a genetic marker for host response to porcine reproductive and respiratory syndrome (PRRS), PRRS vaccination, and co-infection with porcine circovirus type 2b (PCV2b). Objectives of this study were to identify genomic regions other than WUR associated with host response to PRRS vaccination and PRRSV/PCV2b co-infection and regions with a different effect on host response to co-infection, depending on previous vaccination for PRRS. METHODS: Commercial crossbred nursery pigs were pre-selected for WUR genotype (n = 171 AA and 198 AB pigs) where B is the dominant and favorable allele. Half of the pigs were vaccinated for PRRS and 4 weeks later, all pigs were co-infected with PRRS virus and PCV2b. Average daily gain (ADG) and viral load (VL) were quantified post vaccination (Post Vx) and post co-infection (Post Co-X). Single-SNP genome-wide association analyses were then conducted to identify genomic regions associated with response to vaccination and co-infection. RESULTS: Multiple SNPs near the major histocompatibility complex were significantly associated with PCV2b VL (-log 10 P ≥ 5.5), regardless of prior vaccination for PRRS. Several SNPs were also significantly associated with ADG Post Vx and Post Co-X. SNPs with a different effect on ADG, depending on prior vaccination for PRRS, were identified Post Vx (-log 10 P = 5.6) and Post Co-X (-log 10 P = 5.5). No SNPs were significantly associated with vaccination VL (-log10 P ≤ 4.7) or PRRS VL (-log10 P ≤ 4.3). Genes near SNPs associated with vaccination VL, PRRS VL, and PCV2b VL were enriched (P ≤ 0.01) for immune-related pathways and genes near SNPs associated with ADG were enriched for metabolism pathways (P ≤ 0.04). SNPs associated with vaccination VL, PRRS VL, and PCV2b VL showed overrepresentation of health QTL identified in previous studies and SNPs associated with ADG Post Vx of Non-Vx pigs showed overrepresentation of growth QTL. CONCLUSIONS: Multiple genomic regions were associated with PCV2b VL and ADG Post Vx and Post Co-X. Different SNPs were associated with ADG, depending on previous vaccination for PRRS. Results of functional annotation analyses and novel approaches of using previously-reported QTL support the identified regions.

Knockout of maternal CD163 protects fetuses from infection with porcine reproductive and respiratory syndrome virus (PRRSV).

After infection of the porcine dam at about 90 days of gestation, porcine reproductive and respiratory syndrome virus (PRRSV) crosses the placenta and begins to infect fetuses. Outcomes of include abortion, fetal death and respiratory disease in newborn piglets. CD163 is the receptor for the virus. In this study, CD163-positive fetuses, recovered between 109 days of gestation or 20 days after maternal infection, were completely protected from PRRSV in dams possessing a complete knockout of the CD163 receptor. The results demonstrate a practical means to eliminate PRRSV-associated reproductive disease, a major source of economic hardship to agriculture.

T Cell Lymphoma and Leukemia in Severe Combined Immunodeficiency Pigs following Bone Marrow Transplantation: A Case Report.

After the discovery of naturally occurring severe combined immunodeficiency (SCID) within a selection line of pigs at Iowa State University, we found two causative mutations in the Artemis gene: haplotype 12 (ART12) and haplotype 16 (ART16). Bone marrow transplants (BMTs) were performed to create genetically SCID and phenotypically immunocompetent breeding animals to establish a SCID colony for further characterization and research utilization. Of nine original BMT transfer recipients, only four achieved successful engraftment. At approximately 11 months of age, both animals homozygous for the ART16 mutation were diagnosed with T cell lymphoma. One of these ART16/ART16 recipients was a male who received a transplant from a female sibling; the tumors in this recipient consist primarily of Y chromosome-positive cells. The other ART16/ART16 animal also presented with leukemia in addition to T cell lymphoma, while one of the ART12/ART16 compound heterozygote recipients presented with a nephroblastoma at a similar age. Human Artemis SCID patients have reported cases of lymphoma associated with a "leaky" Artemis phenotype. The naturally occurring Artemis SCID pig offers a large animal model more similar to human SCID patients and may offer a naturally occurring cancer model and provides a valuable platform for therapy development.

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.

Tissue localization, shedding, virus carriage, antibody response, and aerosol transmission of Porcine epidemic diarrhea virus following inoculation of 4-week-old feeder pigs.

We determined tissue localization, shedding patterns, virus carriage, antibody response, and aerosol transmission of Porcine epidemic diarrhea virus (PEDV) following inoculation of 4-week-old feeder pigs. Thirty-three pigs were randomly assigned to 1 of 3 groups for the 42-day study: inoculated (group A; n = 23), contact transmission (group B; n = 5), and aerosol transmission (group C; n = 5). Contact transmission occurred rapidly to group B pigs whereas productive aerosol transmission failed to occur to group C pigs. Emesis was the first clinical sign noted at 3 days postinoculation (dpi) followed by mild to moderate diarrhea lasting 5 more days. Real-time PCR detected PEDV in fecal and nasal swabs, oral fluids, serum, and gastrointestinal and lymphoid tissues. Shedding occurred primarily during the first 2 weeks postinoculation, peaking at 5-6 dpi; however, some pigs had PEDV nucleic acid detected in swabs collected at 21 and 28 dpi. Antibody titers were measurable between 14 and 42 dpi. Although feces and intestines collected at 42 dpi were PEDV negative by PCR and immunohistochemistry, respectively, small intestines from 70% of group A pigs were PCR positive. Although disease was relatively mild and transient in this age group, the results demonstrate that 4-week-old pigs are productively infected and can sustain virus replication for several weeks. Long-term shedding of PEDV in subclinically affected pigs should be considered an important source for PEDV transmission.

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.

An intact sialoadhesin (Sn/SIGLEC1/CD169) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus.

Surface expression of SIGLEC1, also known as sialoadhesin or CD169, is considered a primary determinant of the permissiveness of porcine alveolar macrophages for infection by porcine reproductive and respiratory syndrome virus (PRRSV). In vitro, the attachment and internalization of PRRSV are dependent on the interaction between sialic acid on the virion surface and the sialic acid binding domain of the SIGLEC1 gene. To test the role of SIGLEC1 in PRRSV infection, a SIGLEC1 gene knockout pig was created by removing part of exon 1 and all of exons 2 and 3 of the SIGLEC1 gene. The resulting knockout ablated SIGLEC1 expression on the surface of alveolar macrophages but had no effect on the expression of CD163, a coreceptor for PRRSV. After infection, PRRSV viremia in SIGLEC1(-/-) pigs followed the same course as in SIGLEC1(-/+) and SIGLEC1(+/+) littermates. The absence of SIGLEC1 had no measurable effect on other aspects of PRRSV infection, including clinical disease course and histopathology. The results demonstrate that the expression of the SIGLEC1 gene is not required for infection of pigs with PRRSV and that the absence of SIGLEC1 does not contribute to the pathogenesis of acute disease.

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.

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.

Human xenografts are not rejected in a naturally occurring immunodeficient porcine line: a human tumor model in pigs.

Animal models for cancer therapy are invaluable for preclinical testing of potential cancer treatments; however, therapies tested in such models often fail to translate into clinical settings. Therefore, a better preclinical model for cancer treatment testing is needed. Here we demonstrate that an immunodeficient line of pigs can host and support the growth of xenografted human tumors and has the potential to be an effective animal model for cancer therapy. Wild-type and immunodeficient pigs were injected subcutaneously in the left ear with human melanoma cells (A375SM cells) and in the right ear with human pancreatic carcinoma cells (PANC-1). All immunodeficient pigs developed tumors that were verified by histology and immunohistochemistry. Nonaffected littermates did not develop tumors. Immunodeficient pigs, which do not reject xenografted human tumors, have the potential to become an extremely useful animal model for cancer therapy because of their similarity in size, anatomy, and physiology to humans.

Probing genetic control of swine responses to PRRSV infection: current progress of the PRRS host genetics consortium.

BACKGROUND: Understanding the role of host genetics in resistance to porcine reproductive and respiratory syndrome virus (PRRSV) infection, and the effects of PRRS on pig health and related growth, are goals of the PRRS Host Genetics Consortium (PHGC). METHODS: The project uses a nursery pig model to assess pig resistance/susceptibility to primary PRRSV infection. To date, 6 groups of 200 crossbred pigs from high health farms were donated by commercial sources. After acclimation, the pigs were infected with PRRSV in a biosecure facility and followed for 42 days post infection (dpi). Blood samples were collected at 0, 4, 7, 10, 14, 21, 28, 35 and 42 dpi for serum and whole blood RNA gene expression analyses; weekly weights were recorded for growth traits. All data have been entered into the PHGC relational database. Genomic DNAs from all PHGC1-6 pigs were prepared and genotyped with the Porcine SNP60 SNPchip. RESULTS: Results have affirmed that all challenged pigs become PRRSV infected with peak viremia being observed between 4-21 dpi. Multivariate statistical analyses of viral load and weight data have identified PHGC pigs in different virus/weight categories. Sera are now being compared for factors involved in recovery from infection, including speed of response and levels of immune cytokines. Genome-wide association studies (GWAS) are underway to identify genes and chromosomal locations that identify PRRS resistant/susceptible pigs and pigs able to maintain growth while infected with PRRSV. CONCLUSIONS: Overall, the PHGC project will enable researchers to discover and verify important genotypes and phenotypes that predict resistance/susceptibility to PRRSV infection. The availability of PHGC samples provides a unique opportunity to continue to develop deeper phenotypes on every PRRSV infected pig.

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.

Evidence for recombination between PCV2a and PCV2b in the field.

Genomic sequence analysis demonstrates that porcine circovirus type 2 (PCV2) isolates are divided into distinct genotypes. Historically, swine herds in the U.S. have been infected with the PCV2a genotype. In 2005, PCV2b was identified in North America and with it increased reports of porcine circovirus disease (PCVD). A differential PCR technique incorporating PCV2 genotype-specific primers was used in the clinical diagnosis of PCVD. A set of 97 diagnostic submissions showed that both PCV2a and PCV2b were present in 25% of clinical samples. The construction of phylogenetic trees using whole genome sequences from diagnostic submissions showed that one isolate, 0737A, was only loosely associated with other PCV2b isolates. Analysis of the variable sites between representative PCV2a and PCV2b DNA sequences and the 0737A sequence, showed that 0737A was a mosaic sequence, with the ORF1 region from PCV2a and ORF2 from PCV2b. This study demonstrates that pigs can be naturally infected with multiple PCV2 genotypes and that PCV2a/PCV2b recombination events occur in the field.

Expression and stability of foreign tags inserted into nsp2 of porcine reproductive and respiratory syndrome virus (PRRSV).

The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the single largest protein produced during infection. The cDNA of the pCMV-129 infectious PRRSV clone was modified for accepting foreign tags by first creating unique Mlu I and SgrA I restrictions sites at nucleotide (nt) positions 3219 and 3614, respectively, within the C-terminal region of nsp2. cDNAs encoding oligo- and polypeptide tags, including FLAG, enhanced green fluorescent protein (EGFP) and luciferase were inserted into the newly created restriction sites. The results showed that only the EGFP-containing genomes were properly expressed and produced virus. EGFP fluorescence, but not EGFP immunoreactivity, was lost during passage of recombinant EGFP viruses in culture. Sequencing of a fluorescent-negative EGFP virus showed that the EGFP remained intact, except for the appearance of arginine to cysteine mutation at position 96, which may interfere with chromophore formation or function.

Intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in vero cells.

The nucleocapsid (N) protein of several members within the order Nidovirales localizes to the nucleolus during infection and after transfection of cells with N genes. However, confocal microscopy of N protein localization in Vero cells infected with the severe acute respiratory syndrome coronavirus (SARS-CoV) or transfected with the SARS-CoV N gene failed to show the presence of N in the nucleoplasm or nucleolus. Amino acids 369 to 389, which contain putative nuclear localization signal (NLS) and nucleolar localization signal motifs, failed to restore nuclear localization to an NLS-minus mutant Rev protein. These data indicate that nuclear localization is not a conserved property among all nidoviruses.