Ada G. Cino-Ozuna

Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus

VOLUME 34 NUMBER 1 JANUARY 2016 NATURE BIOTECHNOLOGY To the Editor: Porcine reproductive and respiratory syndrome (PRRS) is the most economically important disease of swine in North America, Europe and Asia, costing producers in North America more than

Characterization of a New Disease Syndrome Associated with Porcine Circovirus Type 2 in Previously Vaccinated Herds

600 million annually1. The disease syndrome was first recognized in the United States in 1987 and described in 1989 (ref. 2). The causative agent, porcine reproductive and respiratory syndrome virus (PRRSV), was subsequently isolated and characterized in Europe in 1991 (ref. 3). Vaccines have been unable to control the disease. It has been suggested that CD163 is the receptor for entry of PRRSV into cells4. Thus, we hypothesized that pigs with defective CD163 would be immune to PRRSV. Previously we used CRISPRCas9 to generate pigs lacking functional CD163 (ref. 5). Here we demonstrate that these animals are resistant to the PRRSV isolate NVSL 97-7895, a well-characterized, relatively virulent viral isolate that is commonly used in experimental PRRSV infection trials. After infection, they showed no clinical signs (fever or respiratory signs), lung pathology, viremia or antibody response and remained healthy for the 35 d after infection measured in this study. Because CD163 was edited using CRISPR-Cas9, the pigs challenged in this study do not contain any transgenes5. PRRSV is a member of the mammalian arterivirus group, which also includes murine lactate dehydrogenase-elevating virus, simian hemorrhagic fever virus and equine arteritis virus. The arteriviruses share important pathogenesis properties, including macrophage tropism and the capacity to cause both severe disease and persistent infection. In young pigs, infection with PRRSV results in respiratory disease, including cough and fever and reduced growth performance. In pregnant sows, PRRSV infection can result in reproductive failure, as well as persistently infected and low birth weight piglets.The virus is associated with polymicrobial disease syndromes, including porcine respiratory Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus

Recognition of the Different Structural Forms of the Capsid Protein Determines the Outcome following Infection with Porcine Circovirus Type 2

ABSTRACT Porcine circovirus-associated disease (PCVAD) encompasses a group of wasting syndromes linked to porcine circovirus type 2 (PCV2). This paper describes a new PCV2 disease syndrome, called acute pulmonary edema (APE), which, unlike other PCVAD syndromes, has a peracute onset and is associated with herds vaccinated for PCV2.

Microbiome associations in pigs with the best and worst clinical outcomes following co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2).

ABSTRACT 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.

Analysis of blood leukocytes in a naturally occurring immunodeficiency of pigs shows the defect is localized to B and T cells.

On a world-wide basis, co-infections involving porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are common and contribute to a range of polymicrobial disease syndromes in swine. Both viruses compromise host defenses, resulting in increased susceptibility to infections by primary and secondary pathogens that can affect growth performance as well as increased morbidity and mortality. An experimental population of 95 pigs was co-infected with PRRSV and PCV2. At 70days post-infection (dpi), 20 representative pigs were selected as having the best or worst clinical outcome based on average daily gain (ADG) and the presence of clinical disease. Worst clinical outcome pigs had prolonged and greater levels of viremia as measured by qPCR. Serum, lung and fecal samples collected at 70 dpi were analyzed using a comprehensive DNA microarray technology, the Lawrence Livermore Microbial Detection Array, to detect over 8000 microbes. Bacterial species, such as Bacillus cereus, were detected at a higher rate in the serum of worst performing pigs. At the level of the fecal microbiome, the overall microbial diversity was lower in the worst clinical outcome group. The results reinforce the importance of pathogen load in determining clinical outcome and suggest an important role of microbial diversity as a contributing factor in disease.

Increased microbiome diversity at the time of infection is associated with improved growth rates of pigs after co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2)

Severe combined immunodeficiency (SCID) is the result of a set of inherited genetic defects which render components of the immune response nonfunctional. In Arabian horses, Jack Russell terriers, and mice, the disorder is a consequence of the absence of T and B lymphocytes, while natural killer (NK) cell and other leukocyte populations remain intact. Preliminary analysis of a naturally acquired form of inherited SCID in a line of pigs showed several defects in the architecture and composition of secondary lymphoid organs. In this study, a quantitative assessment of lymphocyte populations in affected and normal littermates showed depleted T or B lymphocyte populations in affected pigs; however, NK cells and neutrophils were present in numbers comparable to unaffected littermates. The results indicate that the immune defect in pigs shares the same features as other SCID-affected species.

Toward the development of a one-dose classical swine fever subunit vaccine: antigen titration, immunity onset, and duration of immunity

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are two of the most important pathogens affecting the swine industry worldwide. Co-infections are common on a global scale, resulting in pork production losses through reducing weight gain and causing respiratory disease in growing pigs. Our initial work demonstrated that the fecal microbiome was associated with clinical outcome of pigs 70days post-infection (dpi) with PRRSV and PCV2. However, it remained uncertain if microbiome characteristics could predispose response to viral infection. The purpose of this study was to determine if microbiome characteristics present at the time of virus exposure were associated with outcome after co-infection. Using the Lawrence Livermore Microbial Detection Array, we profiled the microbiome in feces prior to infection from pigs identified retrospectively as having high or low growth rates after co-infection. High growth rate pigs had less severe interstitial pneumonia, reduced virus replication, and a significant increase in average daily weight gain throughout the study. At the level of the fecal microbiome, high growth rate pigs had increased microbial diversity on both a family and species level. Shifts in the microbiome composition of high growth rate pigs included reduced Methanobacteriaceae species, increased Ruminococcaceae species, and increased Streptococcaceae species when compared to low growth rate pigs. The results indicate that both microbiome diversity and composition at the time of virus exposure may play a role in the subsequent response of pigs to PRRSV/PCV2 co-infection.

Construction of Recombinant Porcine Reproductive and Respiratory Syndrome Virus Expressing CSFV E2 Glycoprotein

Highly contagious classical swine fever (CSF) remains a major trade and health problem in the pig industry, resulting in large economic losses worldwide. In CSF-endemic countries, attenuated CSF virus (CSFV) vaccines have been routinely used to control the disease. However, eradication of CSFV in a geographical area would require permanent reduction to zero presence of the virus. It is therefore of paramount importance to develop a safe, potent, and non-infectious CSF vaccine. We have previously reported on a cost-effective CSF E2 subunit vaccine, KNB-E2, which can protect against CSF symptoms in a single dose containing 75 µg of recombinant CSFV glycoprotein E2. In this study, we report on a series of animal studies undertaken to elucidate further the efficacy of KNB-E2. We found that pigs vaccinated with a single KNB-E2 dose containing 25 µg of recombinant CSFV glycoprotein E2 were protected from clinical symptoms of CSF. In addition, KNB-E2-mediated reduction of CSF symptoms was observed at two weeks post-vaccination and the vaccinated pigs continued to exhibit reduced CSF clinical signs when virus challenged at two months and four months post-vaccination. These results suggest that KNB-E2 effectively reduces CSF clinical signs, indicating the potential of this vaccine for safely minimizing CSF-related losses.

Fecal Microbiota Transplantation Is Associated With Reduced Morbidity and Mortality in Porcine Circovirus Associated Disease

Classical Swine Fever (CSF) and Porcine Reproductive and Respiratory Syndrome (PRRS) are two highly contagious infectious diseases caused by CSF virus (CSFV) and PRRS virus (PRRSV), respectively. Recombinant PRRSV expressing CSFV E2 glycoprotein could be used for the development of bivalent vaccine, antiviral drug or antibody screening assays against PRRSV and CSFV. In this study, a recombinant PRRSV expressing CSFV E2 glycoprotein (p129-CSFV-E2) was constructed. The E2 gene from CSFV C-strain vaccine was cloned and inserted between ORF1b and ORF2 gene of the PRRSV P129 strain. An additional transcriptional regulatory sequence 6 (TRS6) was inserted following the CSFV E2 for driving the transcription of ORF2. The construct efficiently produced progeny viruses and the expressed CSFV E2 protein was detected by immune staining of infected MARC145 cells.The growth ability of the p129-CSFV-E2 virus is comparable to the parental p129 virus. The genetic stability and stable expression of CSFV E2 of P129-CSFV-E2 virus could reach 11 passages in cell culture. The results showed that CSFV E2 glycoprotein could be expressed as a separated subgenomic unit in the PRRSV genome. The recombinant P129-CSFV-E2 virus can be useful for the development of novel vaccines, cell-based high throughput antiviral drug and antibody screening system against PRRSV and CSFV.

Abstract LB-116: Immunodeficient pigs as a large animal model for human tumors

Porcine circovirus associated disease (PCVAD) is a term used to describe the multi-factorial disease syndromes caused by porcine circovirus type 2 (PCV-2), which can be reproduced in an experimental setting through the co-infection of pigs with PCV-2 and porcine reproductive and respiratory syndrome virus (PRRSV). The resulting PCVAD-affected pigs represent a subpopulation within the co-infected group. In co-infection studies, the presence of increased microbiome diversity is linked to a reduction in clinical signs. In this study, fecal microbiota transplantation (FMT) was investigated as a means to prevent PCVAD in pigs co-infected with PRRSV and PCV-2d. The sources of the FMT material were high-parity sows with a documented history of high health status and robust litter characteristics. The analysis of the donated FMT material showed the absence of common pathogens along with the presence of diverse microbial phyla and families. One group of pigs (n = 10) was administered the FMT while a control group (n = 10) was administered a sterile mock-transplant. Over the 42-day post-infection period, the FMT group showed fewer PCVAD-affected pigs, as evidenced by a significant reduction in morbidity and mortality in transplanted pigs, along with increased antibody levels. Overall, this study provides evidence that FMT decreases the severity of clinical signs following co-infection with PRRSV and PCV-2 by reducing the prevalence of PCVAD.