Juan E. Abrahante

The ever-expanding diversity of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) virus appeared 20 years ago as the cause of a new disease in swine. Today PRRS is the most significant swine disease worldwide in spite of intensive immunological interventions. The virus showed remarkable genetic variation with two geographically distinct genotypes at the time of its discovery, indicating the possibility of prolonged evolutionary divergence prior to its appearance as a swine pathogen. Since then, both type 1 and type 2 have spread geographically, radiated genetically, and acquired new phenotypic characteristics, especially increased virulence. Here, we explore various hypotheses that might account for rapid expansion and diversification of PRRSV, including mechanisms specific to PRRSV and other arteriviruses, cellular modification processes, and immunological selection. Phylogenetic analysis of PRRSV has provided a broadly applicable means to relate diverse isolates, but it does not explain biological variation in virulence or immunological cross-protection. We present other methods of classification and review their limitations. Major questions about PRRSV remain unanswered despite intensive investigation, suggesting that the interaction of PRRSV with pigs involves novel biological processes that may be relevant to other RNA virus and host interactions.

Absence of porcine circovirus type 1 (PCV1) and high prevalence of PCV 2 exposure and infection in swine finisher herds

Porcine circovirus (PCV) appeared in 1974 as an unidentified, innocuous viral inhabitant of cell cultures and pigs. Today PCV1 is a contaminant of some human vaccines, and PCV2 is a major pathogen of swine. PCV1 is reportedly ubiquitous in swine but nonpathogenic. Since the interplay of PCV1 and PCV2 in swine might explain variable disease results and shed light on the potential for human exposure, we analyzed in depth the prevalence of PCV1 and PCV2 infection and exposure in the U.S. finishing swine herd. Over 82% of sera from 185 farms were positive for PCV2 by PCR, whereas only 2.4% were positive for PCV1. More than 80% of PCV2 DNA-positive swine were also positive for anti-PCV2 antibodies. PCV1 was only rarely present. Exposure of swine, and therefore humans via pigs, to PCV1 is negligible. We conclude that PCV2 causes a persistent infection in pigs and that PCV1 is absent or rare in swine.

CFTR is a tumor suppressor gene in murine and human intestinal cancer

CFTR, the cystic fibrosis (CF) gene, encodes for the CFTR protein that plays an essential role in anion regulation and tissue homeostasis of various epithelia. In the gastrointestinal (GI) tract CFTR promotes chloride and bicarbonate secretion, playing an essential role in ion and acid–base homeostasis. Cftr has been identified as a candidate driver gene for colorectal cancer (CRC) in several Sleeping Beauty DNA transposon-based forward genetic screens in mice. Further, recent epidemiological and clinical studies indicate that CF patients are at high risk for developing tumors in the colon. To investigate the effects of CFTR dysregulation on GI cancer, we generated ApcMin mice that carried an intestinal-specific knockout of Cftr. Our results indicate that Cftr is a tumor suppressor gene in the intestinal tract as Cftr mutant mice developed significantly more tumors in the colon and the entire small intestine. In Apc+/+ mice aged to ~1 year, Cftr deficiency alone caused the development of intestinal tumors in >60% of mice. Colon organoid formation was significantly increased in organoids created from Cftr mutant mice compared with wild-type controls, suggesting a potential role of Cftr in regulating the intestinal stem cell compartment. Microarray data from the Cftr-deficient colon and the small intestine identified dysregulated genes that belong to groups of immune response, ion channel, intestinal stem cell and other growth signaling regulators. These associated clusters of genes were confirmed by pathway analysis using Ingenuity Pathway Analysis and gene set enrichment analysis (GSEA). We also conducted RNA Seq analysis of tumors from Apc+/+ Cftr knockout mice and identified sets of genes dysregulated in tumors including altered Wnt β-catenin target genes. Finally we analyzed expression of CFTR in early stage human CRC patients stratified by risk of recurrence and found that loss of expression of CFTR was significantly associated with poor disease-free survival.CFTR, the cystic fibrosis (CF) gene, encodes for the CFTR protein that plays an essential role in anion regulation and tissue homeostasis of various epithelia. In the gastrointestinal (GI) tract CFTR promotes chloride and bicarbonate secretion, playing an essential role in ion and acid-base homeostasis. Cftr has been identified as a candidate driver gene for colorectal cancer (CRC) in several Sleeping Beauty DNA transposon-based forward genetic screens in mice. Further, recent epidemiological and clinical studies indicate that CF patients are at high risk for developing tumors in the colon. To investigate the effects of CFTR dysregulation on GI cancer, we generated Apc(Min) mice that carried an intestinal-specific knockout of Cftr. Our results indicate that Cftr is a tumor suppressor gene in the intestinal tract as Cftr mutant mice developed significantly more tumors in the colon and the entire small intestine. In Apc(+/+) mice aged to ~1 year, Cftr deficiency alone caused the development of intestinal tumors in >60% of mice. Colon organoid formation was significantly increased in organoids created from Cftr mutant mice compared with wild-type controls, suggesting a potential role of Cftr in regulating the intestinal stem cell compartment. Microarray data from the Cftr-deficient colon and the small intestine identified dysregulated genes that belong to groups of immune response, ion channel, intestinal stem cell and other growth signaling regulators. These associated clusters of genes were confirmed by pathway analysis using Ingenuity Pathway Analysis and gene set enrichment analysis (GSEA). We also conducted RNA Seq analysis of tumors from Apc(+/+) Cftr knockout mice and identified sets of genes dysregulated in tumors including altered Wnt β-catenin target genes. Finally we analyzed expression of CFTR in early stage human CRC patients stratified by risk of recurrence and found that loss of expression of CFTR was significantly associated with poor disease-free survival.

The Candidate Cancer Gene Database: a database of cancer driver genes from forward genetic screens in mice

Identification of cancer driver gene mutations is crucial for advancing cancer therapeutics. Due to the overwhelming number of passenger mutations in the human tumor genome, it is difficult to pinpoint causative driver genes. Using transposon mutagenesis in mice many laboratories have conducted forward genetic screens and identified thousands of candidate driver genes that are highly relevant to human cancer. Unfortunately, this information is difficult to access and utilize because it is scattered across multiple publications using different mouse genome builds and strength metrics. To improve access to these findings and facilitate meta-analyses, we developed the Candidate Cancer Gene Database (CCGD, http://ccgd-starrlab.oit.umn.edu/). The CCGD is a manually curated database containing a unified description of all identified candidate driver genes and the genomic location of transposon common insertion sites (CISs) from all currently published transposon-based screens. To demonstrate relevance to human cancer, we performed a modified gene set enrichment analysis using KEGG pathways and show that human cancer pathways are highly enriched in the database. We also used hierarchical clustering to identify pathways enriched in blood cancers compared to solid cancers. The CCGD is a novel resource available to scientists interested in the identification of genetic drivers of cancer.

Microbiological safety of porcine islets: comparison with source pig

Abrahante JE, Martins K, Papas KK, Hering BJ, Schuurman H‐J, Murtaugh MP. Microbiological safety of porcine islets: comparison with source pig. Xenotransplantation 2011; 18: 88–93.

Comparative genome analysis of an avirulent and two virulent strains of avian Pasteurella multocida reveals candidate genes involved in fitness and pathogenicity

BackgroundPasteurella multocida is the etiologic agent of fowl cholera, a highly contagious and severe disease of poultry causing significant mortality and morbidity throughout the world. All types of poultry are susceptible to fowl cholera. Turkeys are most susceptible to the peracute/acute forms of the disease while chickens are most susceptible to the acute and chronic forms of the disease. The whole genome of the Pm70 strain of P. multocida was sequenced and annotated in 2001. The Pm70 strain is not virulent to chickens and turkeys. In contrast, strains X73 and P1059 are highly virulent to turkeys, chickens, and other poultry species. In this study, we sequenced the genomes of P. multocida strains X73 and P1059 and undertook a detailed comparative genome analysis with the avirulent Pm70 strain. The goal of this study was to identify candidate genes in the virulent strains that may be involved in pathogenicity of fowl cholera disease.ResultsComparison of virulent versus avirulent avian P. multocida genomes revealed 336 unique genes among the P1059 and/or X73 genomes compared to strain Pm70. Genes of interest within this subset included those encoding an L-fucose transport and utilization system, several novel sugar transport systems, and several novel hemagglutinins including one designated PfhB4. Additionally, substantial amino acid variation was observed in many core outer membrane proteins and single nucleotide polymorphism analysis confirmed a higher dN/dS ratio within proteins localized to the outer membrane.ConclusionsComparative analyses of highly virulent versus avirulent avian P. multocida identified a number of genomic differences that may shed light on the ability of highly virulent strains to cause disease in the avian host, including those that could be associated with enhanced virulence or fitness.

Immune response to ORF5a protein immunization is not protective against Porcine Reproductive and Respiratory Syndrome Virus infection

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus responsible for PRRS in swine, a disease with globally significant animal welfare and economic concerns. There is no specific treatment and variably effective immune protection. Molecular mechanisms responsible for virulence, pathogenesis and protective immune response remain poorly understood. These factors limit progress toward development of effective measures for prevention and treatment of PRRS. A novel PRRSV ORF5a protein, encoded in an open reading frame (ORF) that overlaps the major envelope glycoprotein GP5 ORF, was recently identified. Because ORF5a is highly conserved in diverse PRRSV isolates, is a structural protein in the virion, and elicits a specific antibody response in infected pigs, we investigated its potential role in immune protection against PRRSV infection. Pigs immunized with ORF5a protein had robust serologic responses. However, the antibodies did not neutralize virus, and immunity did not protect against challenge infection. We conclude from these findings that the ORF5a antibody response is neither neutralizing nor protective.

Reverse Transcription Loop-Mediated Isothermal Amplification for the Detection of Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine. The objective of the current study is to investigate the feasibility of using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of PRRSV. The RT-LAMP is a recently described DNA amplification technique reported to be simple, inexpensive, fast, and accurate. The RT-LAMP reaction was set up using 2 sets of primers that were designed to detect North American and European strains of PRRSV and performed successfully in a simple heat block. The specificity of the amplified product was demonstrated by restriction analysis. The RT-LAMP was able to detect 5 different PRRSV isolates. However, the limit of detection ranged between 10 2 and 10 4 50% tissue culture infective dose/ml. The RT-LAMP was further evaluated using serum samples from animals of known infection status. The ability of RT-LAMP to detect PRRSV in serum from acutely infected animals was evaluated with 114 serum samples from 18 experimentally inoculated boars. Forty-nine of these samples tested positive by RT-LAMP, while 94 were positive by reverse transcription polymerase chain reaction (RT-PCR). The diagnostic specificity, evaluated with 100 known negative serum samples, was estimated as 99%. The feasibility of RT-LAMP to detect PRRSV was demonstrated in the current study. The RT-LAMP reaction could be performed in just 1 hr with a simple and inexpensive heat block. However, the sensitivity of this technique was significantly lower than that of RT-PCR.

Presence of Oxalobacter formigenes in the intestinal tract is associated with the absence of calcium oxalate urolith formation in dogs

The incidence of calcium oxalate (CaOx) urolithiasis in dogs has increased steadily over the last two decades. A potential mechanism to minimize CaOx urolithiasis is to reduce enteric absorption of dietary oxalate by oxalate-metabolizing enteric bacteria. Enteric colonization of Oxalobacter formigenes, an anaerobe which exclusively relies on oxalate metabolism for energy, is correlated with absence of hyperoxaluria or CaOx urolithiasis or both in humans and laboratory animals. We thus hypothesized that decreased enteric colonization of O. formigenes is a risk factor for CaOx urolithiasis in dogs. Fecal samples from dogs with CaOx uroliths, clinically healthy, age-, breed- and gender-matched dogs, and healthy non-stone forming breed dogs were screened for the presence of O. formigenes by quantitative PCR to detect the oxalyl CoA decarboxylase (oxc) gene, and by oxalate degrading biochemical activity in fecal cultures. Prevalence of O. formigenes in dogs with CaOx uroliths was 25%, compared to 50% in clinically healthy, age-, breed- and gender-matched dogs, and 75% in healthy non-stone forming breeds. The presence of oxc genes of O. formigenes was significantly higher in healthy non-stone forming breed dogs than in the dogs with CaOx stones. Further, dogs with calcium oxalate stones and the stone-forming breed-matched controls showed comparable levels of biochemical oxalate degrading activity. We conclude that the absence of enteric colonization of O. formigenes is a risk factor for CaOx urolithiasis.

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.