Introductory Paragraph Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel virus that emerged in 2012, causing acute respiratory distress syndrome (ARDS), severe pneumonia-like symptoms, and multi-organ failure, with a case fatality rate of ~36%. Limited clinical studies indicate that humans infected with MERS-CoV exhibited pathology consistent with late stages of ARDS, which is reminiscent of disease observed in patients infected with SARS coronavirus. Models of MERS-CoV-induced severe respiratory disease have been difficult to achieve, and small animal models traditionally used to investigate viral pathogenesis (mouse, hamster, guinea pig, and ferret) are naturally resistant to MERS-CoV. Therefore, we used CRISPR/Cas9 to modify the mouse genome to encode two human amino acids (288 and 330) in the dipeptidyl peptidase 4 receptor, making mice susceptible to MERS-CoV replication. Serial MERS-CoV passage in these engineered mice was then used to generate a mouse-adapted virus that replicated efficiently within the lungs, and evoked symptoms indicative of severe acute respiratory distress syndrome (ARDS), including decreased survival, extreme weight loss, decreased pulmonary function, pulmonary hemorrhage, and pathological signs indicative of end stage lung disease. Importantly, therapeutic countermeasures comprising MERS-CoV neutralizing antibody treatment or a MERS-CoV spike protein vaccine protected engineered mice against MERS-CoV-induced ARDS.

The capacity to efficiently transduce nondividing cells, shuttle large genetic payloads, and maintain stable long-term transgene expression are attributes that have brought lentiviral vectors to the forefront of gene delivery vehicles for research and therapeutic applications in a clinical setting. Our discussion initiates with advances in lentiviral vector development and how these sophisticated lentiviral vectors reflect improvements in safety, regarding the prevention of replication competent lentiviruses (RCLs), vector mobilization, and insertional mutagenesis. Additionally, we describe conventional molecular regulatory systems to manage gene expression levels in a spatial and temporal fashion in the context of a lentiviral vector. State of the art technology for lentiviral vector production by transient transfection and packaging cell lines are explicitly presented with current practices used for concentration, purification, titering, and determining the safety of a vector stock. We summarize lentiviral vector applications that have received a great deal of attention in recent years including the generation of transgenic animals and the stable delivery of RNA interference molecules. Concluding remarks address some of the successes in preclinical animals, and the recent transition of lentiviral vectors to human clinical trials as therapy for a variety of infectious and genetic diseases.

Nitric oxide (NO) is known to be an important endogenous modulator of leukocyte-endothelial cell interactions within the microcirculation. We examined leukocyte rolling and adhesion under baseline conditions and following thrombin (0.25 U/ml) superfusion in the mesentery of wild-type, inducible NOS (iNOS)-deficient (−/−), neuronal NOS (nNOS) −/−, and endothelial cell NOS (ecNOS) −/− mice to further our understanding of NO and leukocyte function. Baseline leukocyte rolling (cells/min) was significantly elevated in both the nNOS −/− (30.0 ± 4.0) and ecNOS −/− mice (67.0 ± 12.0) compared with wild-type mice (11.0 ± 1.4). In addition, baseline leukocyte adherence (cells/100 μm of vessel) was also significantly elevated in the nNOS −/− (5.2 ± 1.0) and ecNOS −/− (13.0 ± 1.3) compared with wild-type animals (1.3 ± 0.5). Deficiency of iNOS had no effect on baseline leukocyte rolling or adhesion in the mesentery. Baseline surface expression of P-selectin was observed in 68.0 ± 9.0% of intestinal venules in ecNOS −/− mice compared with 10.0 ± 2.0% in wild-type mice. Additional studies demonstrated that administration of an anti-P-selectin monoclonal antibody (RB40.34) or the soluble P-selectin ligand, PSGL-1, completely inhibited the increased rolling and firm adhesion response in nNOS −/− and ecNOS −/− mice. Transmigration of neutrophils into the peritoneum following thioglycollate injection was also significantly augmented in nNOS −/− and ecNOS −/− mice. These studies clearly indicate the NO derived from both nNOS and ecNOS is critical in the regulation of leukocyte-endothelial cell interactions.

Human dipeptidyl peptidase 4 (hDPP4) was recently identified as the receptor for Middle East respiratory syndrome coronavirus (MERS-CoV) infection, suggesting that other mammalian DPP4 orthologs may also support infection. We demonstrate that mouse DPP4 cannot support MERS-CoV infection. However, employing mouse DPP4 as a scaffold, we identified two critical amino acids (A288L and T330R) that regulate species specificity in the mouse. This knowledge can support the rational design of a mouse-adapted MERS-CoV for rapid assessment of therapeutics. Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently identified betacoronavirus that can infect the lower respiratory airway of humans, leading to acute respiratory distress syndrome (ARDS) with ϳ43% mortality in hospitalized individuals (1). Disease symptoms associated with MERS-CoV are similar to those of severe acute respiratory syndrome coronavirus (SARS-CoV); however, MERS-CoV is phylogenetically more closely related to the bat coronaviruses HKU4 and HKU5 (2, 3). MERS-CoV also differs from SARS-CoV in terms of receptor usage, where MERS-CoV utilizes dipeptidyl peptidase 4 (DPP4) as an entry receptor (4). Given the importance of MERS-CoV as an emerging pathogen, there is a clear need for the development of new therapeutics, which requires the appropriate animal models. However, to date, nonhuman primates are the only reported animal model for MERS-CoV replication, while traditional small animal models, such as ferrets (5), hamsters (6), and mice (7), are nonpermissive. Given that species-specific differences in DPP4 may confound animal model development, it is important to identify determinants in DPP4 that govern MERS-CoV host range. Knowledge of DPP4 determinants may provide novel insights into interactions between DPP4 and MERS-CoV spike receptor binding domain (RBD), as well as support development of new small animal models.To test whether mouse DPP4 (mDPP4) is capable of acting as an entry receptor for MERS-CoV, we compared mDPP4 with human DPP4 (hDPP4). An ectopic expression system was utilized to constitutively express mDPP4 and hDPP4 in human embryonic kidney 293T (HEK 293T) cells, which lack detectable expression of endogenous hDPP4 (data not shown). Human DPP4 and mouse DPP4 were expressed either as full-length proteins or as fusions to the Venus protein at the carboxy terminus. HEK 293T cells were transfected with 3 g of the indicated DPP4 expression plasmid, and at ϳ20 h posttransfection, cells were infected at a multiplicity of infection (MOI) of 5 with a recombinant MERSCoV strain designed to express tomato red fluorescent protein (rMERS-CoV-red) (Fig. 1). The rMERS-CoV-red virus is derived from the EMC2012 substrain and was previously shown to infect and replicate in a manner similar to wild-type MERS virus (8). Transfection of the DPP4-Venus fusion constructs resulted in high transfection efficiency (nearly 100%) (Fig. 1A and B). Control HEK 293T cells were poorly permissive for MERS-CoV, while cells overexpressing hDPP4 were readily ...