Clinically available immunosuppression averts rejection but not systemic inflammation after porcine islet xenotransplant in cynomolgus macaques.

A safe, efficacious, and clinically applicable immunosuppressive regimen is necessary for islet xenotransplantation to become a viable treatment option for diabetes. We performed intraportal transplants of wild-type adult porcine islets in 25 streptozotocin-diabetic cynomolgus monkeys. Islet engraftment was good in 21, partial in 3, and poor in 1 recipient. Median xenograft survival was 25 days with rapamycin and CTLA4Ig immunosuppression. Adding basiliximab induction and maintenance tacrolimus to the base regimen significantly extended median graft survival to 147 days (p < .0001), with three animals maintaining insulin-free xenograft survival for 265, 282, and 288 days. We demonstrate that this regimen suppresses non-Gal anti-pig antibody responses, circulating effector memory T cell expansion, effector function, and infiltration of the graft. However, a chronic systemic inflammatory state manifested in the majority of recipients with long-term graft survival indicated by increased neutrophil to lymphocyte ratio, IL-6, MCP-1, CD40, and CRP expression. This suggests that this immunosuppression regimen fails to regulate innate immunity and resulting inflammation is significantly associated with increased incidence and severity of adverse events making this regimen unacceptable for translation. Additional studies are needed to optimize a maintenance regimen for regulating the innate inflammatory response.

Isolation of Porcine Reproductive and Respiratory Syndrome Virus GP5-Specific, Neutralizing Monoclonal Antibodies From Hyperimmune Sows.

Porcine reproductive and respiratory syndrome (PRRS) is a devastating disease which impacts the pig industry worldwide. The disease is caused by PRRS viruses (PRRSV-1 and -2) which leads to abortions and other forms of reproductive failure in sows and severe respiratory disease in growing pigs. Current PRRSV vaccines provide limited protection; only providing complete protection against closely related strains. The development of improved PRRSV vaccines would benefit from an increased understanding of epitopes relevant to protection, including those recognized by antibodies which possess the ability to neutralize distantly related strains. In this work, a reverse vaccinology approach was taken; starting first with pigs known to have a broadly neutralizing antibody response and then investigating the responsible B cells/antibodies through the isolation of PRRSV neutralizing monoclonal antibodies (mAbs). PBMCs were harvested from pigs sequentially exposed to a modified-live PRRSV-2 vaccine as well as divergent PRRSV-2 field isolates. Memory B cells were immortalized and a total of 5 PRRSV-specific B-cell populations were isolated. All identified PRRSV-specific antibodies were found to be broadly binding to all PRRSV-2 isolates tested, but not PRRSV-1 isolates. Antibodies against GP5 protein, commonly thought to possess a dominant PRRSV neutralizing epitope, were found to be highly abundant, as four out of five B cells populations were GP5 specific. One of the GP5-specific mAbs was shown to be neutralizing but this was only observed against homologous and not heterologous PRRSV strains. Further investigation of these antibodies, and others, may lead to the elucidation of conserved neutralizing epitopes that can be exploited for improved vaccine design and lays the groundwork for the study of broadly neutralizing antibodies against other porcine pathogens.

Serum cytokine profiles in healthy nonhuman primates are blunted by sedation and demonstrate sexual dimorphism as detected by a validated multiplex immunoassay.

Cytokine profiling is a valuable tool for monitoring immune responses associated with disease and treatment. This study assessed the impact of sex and sedation on serum cytokines in healthy nonhuman primates (NHPs). Twenty-three cytokines were measured from serum using a bead-based multiplex assay. Assay validation for precision, sensitivity, recovery, linearity, and stability was performed. Samples from male and female cynomolgus and rhesus macaques either cooperating or sedated were compared. All cytokines except TNFα demonstrated acceptable sensitivity and precision, with variable recovery and linearity. IFNγ, IL-2, IL-5, IL-6, IL-8, IL-12/23 (p40), IL-13, IL-15, MCP-1, TGFα, VEGF met acceptance criteria; G-CSF, IL-4, IL-10, MIP1α, sCD40L were marginal. Higher cytokine levels were observed in females and cytokine levels were blunted in sedated NHPs when compared to awake cooperating NHPs. Significant differences observed in cytokines related to sex, species, or imposed by handling highlight the importance of model design on translational relevance for clinical settings.

Characterization of Emerging Swine Viral Diseases through Oxford Nanopore Sequencing Using Senecavirus A as a Model.

Emerging viral infectious diseases present a major threat to the global swine industry. Since 2015, Senecavirus A (SVA) has been identified as a cause of vesicular disease in different countries and is considered an emerging disease. Despite the growing concern about SVA, there is a lack of preventive and diagnostic strategies, which is also a problem for all emerging infectious diseases. Using SVA as a model, we demonstrated that Oxford Nanopore MinION sequencing could be used as a robust tool for the investigation and surveillance of emerging viral diseases. Our results identified that MinION sequencing allowed for rapid, unbiased pathogen detection at the species and strain level for clinical cases. SVA whole genome sequences were generated using both direct RNA sequencing and PCR-cDNA sequencing methods, with an optimized consensus accuracy of 94% and 99%, respectively. The advantages of direct RNA sequencing lie in its shorter turnaround time, higher analytical sensitivity and its quantitative relationship between input RNA and output sequencing reads, while PCR-cDNA sequencing excelled at creating highly accurate sequences. This study developed whole genome sequencing methods to facilitate the control of SVA and provide a reference for the timely detection and prevention of other emerging infectious diseases.

Leveraging a Validated in silico Approach to Elucidate Genotype-Specific VP7 Epitopes and Antigenic Relationships of Porcine Rotavirus A.

Rotavirus A (RVA) remains one of the most widespread causes of diarrheal disease and mortality in piglets despite decades of research and efforts to boost lactogenic immunity for passive protection. Genetic changes at B cell epitopes (BCEs) may be driving failure of lactogenic immunity, which relies on production of IgA antibodies to passively neutralize RVA within the piglet gut, yet little research has mapped epitopes to swine-specific strains of RVA. Here we describe a bioinformatic approach to predict BCEs on the VP7 outer capsid protein using sequence data alone. We first validated the approach using a previously published dataset of VP7-specific cross-neutralization titers, and found that amino acid changes at predicted BCEs on the VP7 protein allowed for accurate recapitulation of antigenic relationships among the strains. Applying the approach to a dataset of swine RVA sequences identified 9 of the 11 known BCEs previously mapped to swine strains, indicating that epitope prediction can identify sites that are known to drive neutralization escape in vitro. Additional genotype-specific BCEs were also predicted that may be the cause of antigenic differences among strains of RVA on farms and should be targeted for further confirmatory work. The results of this work lay the groundwork for high throughput, immunologically-relevant analysis of swine RVA sequence data, and provide potential sites that can be targeted with vaccines to reduce piglet mortality and support farm health.

Establishment and characterization of a porcine B cell lymphoma cell line.

The lack of available, well characterized, established, domestic porcine cell lines hinders the advancement of porcine cellular immunology. A case of multicentric lymphoma was diagnosed in a market weight pig at the time of slaughter. Affected lymph nodes and spleen were collected and used for single cell isolation and analysis. Cell lines were established by 3 rounds of limiting dilution from splenic and subiliac lymph node lymphomas. Surface marker staining identified the cells as CD21+, CD79a+, CD20+, PAX5+, and CD3- and cells were grown and easily passaged in cell culture. Transcriptome analysis was carried out to further characterize these rapidly proliferating cells validating the initial cytometric findings, confirming their identity as B cell lymphomas, and suggesting that they arose from germinal center centroblasts with aberrant control of BCL6 expression. Functional analysis identified the cells as being involved in cancer, cell movement, cell survival, and apoptosis. These new porcine B cell lymphoma cell lines will be a valuable resource for more in-depth cellular investigations into the porcine immune system and cancer, as well as providing a potential tool for the growth of lymphotropic viruses of pigs and humans.

The PRRSV-Specific Memory B Cell Response Is Long-Lived in Blood and Is Boosted During Live Virus Re-exposure.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine health and well-being worldwide largely due to an insufficient understanding of the adaptive immune response to infection leading to ineffective PRRSV control. The memory and anamnestic response to infection are critical gaps in knowledge in PRRSV immunity. The lack of effective tools for the evaluation of the memory response previously hindered the ability to effectively characterize the porcine memory response to infection. However, the creation and validation of a PRRSV nsp7-specific B cell tetramer now facilitates the ability to detect very rare memory B cells and thus define the memory response of the pig. Here, we describe the PRRSV nsp7-specific B cell response following vaccination and challenge in six key secondary lymphoid organs including the identification of PBMCs as the tissue of interest for the memory immune response in pigs. Following live virus challenge of immune animals, an anamnestic response of nsp7-specific memory B cells and neutralizing antibodies was observed. This characterization of the functional humoral immune response to PRRSV answers key questions involved in regional specialization of the immune response following intramuscular inoculation of PRRSV MLV.

MinION sequencing of Streptococcus suis allows for functional characterization of bacteria by multilocus sequence typing and antimicrobial resistance profiling.

In recent years, high-throughput sequencing has revolutionized disease diagnosis by its powerful ability to provide high resolution genomic information. The Oxford Nanopore MinION sequencer has unparalleled potential as a rapid disease diagnostic tool due to its high mobility, accessibility, and short turnaround time. However, there is a lack of rigorous quality assessment and control processes standardizing the testing on the MinION, which is necessary for incorporation into a diagnostic workflow. Thus, our study examined the use of the MinION sequencer for bacterial whole genome generation and characterization. Using Streptococcus suis as a model, we optimized DNA isolation and treatments to be used for MinION sequencing and standardized de novo assembly to quickly generate a full-length consensus sequence achieving a 99.4% average accuracy. The consensus genomes from MinION sequencing were able to accurately predict the multilocus sequence type in 8 out of 10 samples and identified antimicrobial resistance profiles for 100% of the samples, despite the concern of a high error rate. The inability to unequivocally predict sequence types was due to difficulty in differentiating high identity alleles, which was overcome by applying additional error correction methods to increase consensus accuracy. This manuscript provides methods for the use of MinION sequencing for identification of S. suis genome sequence, sequence type, and antibiotic resistance profile that can be used as a framework for identification and classification of other pathogens.

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools.

Prompt detection and effective control of porcine reproductive and respiratory syndrome virus (PRRSV) during outbreaks is important given its immense adverse impact on the swine industry. However, the diagnostic process can be challenging due to the high genetic diversity and high mutation rate of PRRSV. A diagnostic method that can provide more detailed genetic information about pathogens is urgently needed. In this study, we evaluated the ability of Oxford Nanopore MinION direct RNA sequencing to generate a PRRSV whole genome sequence and detect and discriminate virus at the strain-level. A nearly full length PRRSV genome was successfully generated from raw sequence reads, achieving an accuracy of 96% after consensus genome generation. Direct RNA sequencing reliably detected the PRRSV strain present with an accuracy of 99.9% using as few as 5 raw sequencing reads and successfully differentiated multiple co-infecting strains present in a sample. In addition, PRRSV strain information was obtained from clinical samples containing 104 to 106 viral copies or more within 6 hours of sequencing. Overall, direct viral RNA sequencing followed by bioinformatic analysis proves to be a promising approach for identification of the viral strain or strains involved in clinical infections, allowing for more precise prevention and control strategies during PRRSV outbreaks.

Temporal Dynamics of Co-circulating Lineages of Porcine Reproductive and Respiratory Syndrome Virus.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the most important endemic pathogen in the U.S. swine industry. Despite control efforts involving improved biosecurity and different vaccination protocols, the virus continues to circulate and evolve. One of the foremost challenges in its control is high levels of genetic and antigenic diversity. Here, we quantify the co-circulation, emergence and sequential turnover of multiple PRRSV lineages in a single swine-producing region in the United States over a span of 9 years (2009-2017). By classifying over 4,000 PRRSV sequences (open-reading frame 5) into phylogenetic lineages and sub-lineages, we document the ongoing diversification and temporal dynamics of the PRRSV population, including the rapid emergence of a novel sub-lineage that appeared to be absent globally pre-2008. In addition, lineage 9 was the most prevalent lineage from 2009 to 2010, but its occurrence fell to 0.5% of all sequences identified per year after 2014, coinciding with the emergence or re-emergence of lineage 1 as the dominant lineage. The sequential dominance of different lineages, as well as three different sub-lineages within lineage 1, is consistent with the immune-mediated selection hypothesis for the sequential turnover in the dominant lineage. As host populations build immunity through natural infection or vaccination toward the most common variant, this dominant (sub-) lineage may be replaced by an emerging variant to which the population is more susceptible. An analysis of patterns of non- synonymous and synonymous mutations revealed evidence of positive selection on immunologically important regions of the genome, further supporting the potential that immune-mediated selection shapes the evolutionary and epidemiological dynamics for this virus. This has important implications for patterns of emergence and re-emergence of genetic variants of PRRSV that have negative impacts on the swine industry. Constant surveillance on PRRSV occurrence is crucial to a better understanding of the epidemiological and evolutionary dynamics of co-circulating viral lineages. Further studies utilizing whole genome sequencing and exploring the extent of cross-immunity between heterologous PRRS viruses could shed further light on PRRSV immunological response and aid in developing strategies that might be able to diminish disease impact.

Establishment of Systems to Enable Isolation of Porcine Monoclonal Antibodies Broadly Neutralizing the Porcine Reproductive and Respiratory Syndrome Virus.

The rapid evolution of porcine reproductive and respiratory syndrome viruses (PRRSV) poses a major challenge to effective disease control since available vaccines show variable efficacy against divergent strains. Knowledge of the antigenic targets of virus-neutralizing antibodies that confer protection against heterologous PRRSV strains would be a catalyst for the development of next-generation vaccines. Key to discovering these epitopes is the isolation of neutralizing monoclonal antibodies (mAbs) from immune pigs. To address this need, we sought to establish systems to enable the isolation of PRRSV neutralizing porcine mAbs. We experimentally produced a cohort of immune pigs by sequential challenge infection with four heterologous PRRSV strains spanning PRRSV-1 subtypes and PRRSV species. Whilst priming with PRRSV-1 subtype 1 did not confer full protection against a subsequent infection with a PRRSV-1 subtype 3 strain, animals were protected against a subsequent PRRSV-2 infection. The infection protocol resulted in high serum neutralizing antibody titers against PRRSV-1 Olot/91 and significant neutralization of heterologous PRRSV-1/-2 strains. Enriched memory B cells isolated at the termination of the study were genetically programmed by transduction with a retroviral vector expressing the Bcl-6 transcription factor and the anti-apoptotic Bcl-xL protein, a technology we demonstrated efficiently converts porcine memory B cells into proliferating antibody-secreting cells. Pools of transduced memory B cells were cultured and supernatants containing PRRSV-specific antibodies identified by flow cytometric staining of infected MARC-145 cells and in vitro neutralization of PRRSV-1. Collectively, these data suggest that this experimental system may be further exploited to produce a panel of PRRSV-specific mAbs, which will contribute both to our understanding of the antibody response to PRRSV and allow epitopes to be resolved that may ultimately guide the design of immunogens to induce cross-protective immunity.

Characterization of age-related susceptibility of macrophages to porcine reproductive and respiratory syndrome virus.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the most economically important disease affecting swine production worldwide. The severity and susceptibility of PRRSV infection varies with age. Nursery pigs have been shown to be more susceptible to PRRSV infection and a more severe and prolonged infection is observed as compared to growing or adult pigs. However, antibody responses to PRRSV are observed independent of age. Swine are the only known hosts of PRRSV, infection is restricted to cells of monocytic lineage, and fully differentiated porcine alveolar macrophages are the primary target of natural infection. Pulmonary intravascular macrophages from young pigs have been shown to be more susceptible to infection than those from adult pigs. A better understanding of why young pigs and macrophages from young pigs are more susceptible to PRRSV infection is critical to identify mechanisms of infection that can be explored for enhanced treatment or prevention of disease. This study examined PRRSV susceptibility of porcine alveolar macrophages isolated from the lungs of pigs of different age groups, and the presence of cell surface receptors to determine if differences correlated with infection level. The younger the pigs were, the more susceptible the macrophage were to PRRSV infection, but no differences in cellular receptor expression were observed between pigs of different ages. Resistance to infection is likely related to intracellular innate immune mechanisms rather than receptor-mediated entry.

Associations Between Nutrition, Gut Microbiome, and Health in A Novel Nonhuman Primate Model.

Red-shanked doucs (Pygathrix nemaeus) are endangered, foregut-fermenting colobine primates which are difficult to maintain in captivity. There are critical gaps in our understanding of their natural lifestyle, including dietary habits such as consumption of leaves, unripe fruit, flowers, seeds, and other plant parts. There is also a lack of understanding of enteric adaptations, including their unique microflora. To address these knowledge gaps, we used the douc as a model to study relationships between gastrointestinal microbial community structure and lifestyle. We analyzed published fecal samples as well as detailed dietary history from doucs with four distinct lifestyles (wild, semi-wild, semi-captive, and captive) and determined gastrointestinal bacterial microbiome composition using 16S rRNA sequencing. A clear gradient of microbiome composition was revealed along an axis of natural lifestyle disruption, including significant associations with diet, biodiversity, and microbial function. We also identified potential microbial biomarkers of douc dysbiosis, including Bacteroides and Prevotella, which may be related to health. Our results suggest a gradient-like shift in captivity causes an attendant shift to severe gut dysbiosis, thereby resulting in gastrointestinal issues.

Porcine circovirus 2 infection induces IFNß expression through increased expression of genes involved in RIG-I and IRF7 signaling pathways.

Porcine circovirus-associated disease (PCVAD), caused by porcine circovirus 2 (PCV2), is characterized by a highly variable pathogenesis that is manifested by various disease syndromes and includes immune evasion. Hence, even though PCVAD is effectively controlled by vaccination, pigs and farms remain infected so that continued vaccination is necessary to control disease. We investigated the molecular interactions of PCV2 and its permissive VR1BL host cell for gene expression signatures that could provide insight into mechanisms leading towards disease. Molecular pathways involved in the innate immune response to PCV2 infection were examined to identify changes in gene expression associated with productive infection of VR1BL cells. RNA profiling from infected and uninfected cells showed that 139 genes were induced by infection and 43 genes were down-regulated, using a p value <0.05 and an absolute fold-change difference>2. A strong type 1 interferon response, including an increase in genes involved in the RIG-I/MDA5 pathway and downstream interferon induced genes, was observed. Key regulators involved in PCV2 infection were identified as IFNß, DDX58 (RIG-I), and IRF7. PCV2 infection induces a strong interferon response which unexpectedly facilitates viral gene expression, perhaps due to the presence of an interferon-sensitive response element in the viral promoter. The findings suggest that PCV2 interventions that attenuate type 1 interferon responses at the cellular level might enhance immunity and eliminate persistent infection.

Porcine reproductive and respiratory syndrome virus neutralizing antibodies provide in vivo cross-protection to PRRSV1 and PRRSV2 viral challenge.

Vaccine control and prevention of porcine reproductive and respiratory syndrome (PRRS), the most important disease of swine, is difficult to achieve. However, the discovery of broadly neutralizing antibody activity against porcine reproductive and respiratory syndrome virus (PRRSV) under typical field conditions opens the door to new immunologic approaches for robust protection. We show here that passive administration of purified immunoglobulins with neutralizing antibodies reduced PRRSV2 infection by up to 96%, and PRRSV1 infection by up to 87%, whereas immune immunoglobulins lacking neutralizing activity had no effect on viral infection. Hence, immune competence of passive immunoglobulin transfer was associated specifically with antibody neutralizing activity. Current models of PRRSV infection implicate a minor envelope glycoprotein (GP) complex including GP2, GP3, and GP4, as critical to permissive cell infection. However, conserved peptides comprising the putative cell attachment structure did not attenuate neutralization or viral infection. The results show that immunological approaches aimed at induction of broadly neutralizing antibodies may substantially enhance immune protection against PRRSV. The findings further show that naturally occurring viral isolates are able to induce protective humoral immunity against unrelated PRRSV challenge, thus removing a major conceptual barrier to vaccine development.

B Cell Tetramer Development for Veterinary Vaccinology.

Immunological memory is elicited after either vaccination or natural exposure to a pathogen and is essential for protection against re-exposure. Despite its critical importance, the ability to interrogate the veterinary animal memory immune response has long been hindered by a paucity of tools to assess immunological memory. As a result, the evaluation and analysis of protective immune responses that predict immune protection in food and fiber animals and facilitate vaccine development are obstructed. To fill this gap in knowledge in swine, we created a B cell tetramer to porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 7 (nsp7) to efficiently and effectively investigate the memory B cell response, a hallmark of anti-viral immunity. This novel reagent was validated by using a modified capture ELISA, tetramer pulldowns, and flow cytometry, and it was shown to detect rare, antigen-specific B cells that were present at a frequency of about 0.001% of total B lymphocytes in immune animals. The nsp7-B cell tetramer will help to characterize the PRRSV-specific memory B cell response, which is fundamentally important for understanding immunological competence and animal variation in resistance to PRRSV infection. We expect that the method will be widely applicable to the exploration of immunity to veterinary pathogens.

Effect of Maternal Antibody Transfer on Antibody Dynamics and Control of Porcine Circovirus Type 2 Infection in Offspring.

Sow immunity plays an important role in preventing viral infection and disease in newborn piglets. Vertical transmission of porcine circovirus type 2 (PCV2) may perpetuate porcine circovirus associated disease (PCVAD) in newborn and growing pigs. Hence, the immunological effects of maternal immunoglobulin transfer of PCV2-specific antibodies on PCV2 viremia and immune response in piglets in commercial swine herds were evaluated. Sow vaccination has been shown to reduce viral shedding and viremia, and increases the neutralizing antibody (NA) titers. Since NAs are important for control of PCVAD and mammary secretions may contain high anti-PCV2 NA levels, we examined the PCV2 NA levels in colostrum, milk, sow serum, and piglet serum over time to investigate an association between NA levels and protection against infection. NA titers were remarkably high (up to 10-6 50% neutralizing titer) in sow serum and colostrum on all farms regardless of viremia levels. In piglets vaccinated at 3 weeks of age, NA titers peaked at 10 weeks of age and continued to maintain high viral neutralizing titers to slaughter. The impact of maternally derived neutralizing activity was most evident during the suckling period. Although PCV2 was transmitted from sows to piglets in colostrum, piglets were largely nonviremic at weaning. Thus, NAs appear to control or suppress initial infection even though they are unable to clear or prevent infection later in life.

Effect of Booster Vaccination with Inactivated Porcine Epidemic Diarrhea Virus on Neutralizing Antibody Response in Mammary Secretions.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, dehydration in pigs, and high mortality rates in piglets <3 weeks of age. Maternal immunity protects piglets, but information on vaccination before or after natural infection in endemically exposed sow herds is limited. Currently, the recovery goal in sow units infected with PEDV is to become fully naive again or use natural virus infection to develop immune gilts through a feedback program before introduction into the sow herd. Since neutralizing antibodies in the gut are critical for protection against enteric viral infections such as PEDV, we evaluated the effect of a conditionally licensed, adjuvanted inactivated PEDV vaccine on neutralizing antibody levels in milk and colostrum in both naive and previously naturally exposed sow herds. The results illustrate that intramuscular vaccination increased neutralizing antibody titers, and anti-PEDV IgA and IgG in milk and colostrum of sows that were previously infected. Thus, inactivated PEDV vaccines may provide increased protection to piglets nursing on previously infected sows against exposure to PEDV through increased delivery of lactogenic neutralizing antibodies to the enteric site of infection.

Longitudinal Surveillance of Porcine Rotavirus B Strains from the United States and Canada and In Silico Identification of Antigenically Important Sites.

Rotavirus B (RVB) is an important swine pathogen, but control and prevention strategies are limited without an available vaccine. To develop a subunit RVB vaccine with maximal effect, we characterized the amino acid sequence variability and predicted antigenicity of RVB viral protein 7 (VP7), a major neutralizing antibody target, from clinically infected pigs in the United States and Canada. We identified genotype-specific antigenic sites that may be antibody neutralization targets. While some antigenic sites had high amino acid functional group diversity, nine antigenic sites were completely conserved. Analysis of nucleotide substitution rates at amino acid sites (dN/dS) suggested that negative selection appeared to be playing a larger role in the evolution of the identified antigenic sites when compared to positive selection, and was identified in six of the nine conserved antigenic sites. These results identified important characteristics of RVB VP7 variability and evolution and suggest antigenic residues on RVB VP7 that are negatively selected and highly conserved may be good candidate regions to include in a subunit vaccine design due to their tendency to remain stable.

Team science and the physician-scientist in the age of grand health challenges.

Despite remarkable advances in medical research, clinicians face daunting challenges from new diseases, variations in patient responses to interventions, and increasing numbers of people with chronic health problems. The gap between biomedical research and unmet clinical needs can be addressed by highly talented interdisciplinary investigators focused on translational bench-to-bedside medicine. The training of talented physician-scientists comfortable with forming and participating in multidisciplinary teams that address complex health problems is a top national priority. Challenges, methods, and experiences associated with physician-scientist training and team building were explored at a workshop held at the Second International Conference on One Medicine One Science (iCOMOS 2016), April 24-27, 2016, in Minneapolis, Minnesota. A broad range of scientists, regulatory authorities, and health care experts determined that critical investments in interdisciplinary training are essential for the future of medicine and healthcare delivery. Physician-scientists trained in a broad, nonlinear, cross-disciplinary manner are and will be essential members of science teams in the new age of grand health challenges and the birth of precision medicine. Team science approaches have accomplished biomedical breakthroughs once considered impossible, and dedicated physician-scientists have been critical to these achievements. Together, they translate into the pillars of academic growth and success.