MiRNAs from the Dlk1-Dio3 locus and miR-224/452 cluster contribute to glioblastoma tumor heterogeneity.

Glioblastoma is one of the most common and aggressive brain tumors and has seen few improvements in patient outcomes. Inter-tumor heterogeneity between tumors of different patients as well as intra-tumor heterogeneity of cells within the same tumor challenge the development of effective drugs. MiRNAs play an essential role throughout the developing brain and regulate many key genes involved in oncogenesis, yet their role in driving many of the processes underlying tumor heterogeneity remains unclear. In this study, we highlight miRNAs from the Dlk1-Dio3 and miR-224/452 clusters which may be expressed cell autonomously and have expression that is associated with cell state genes in glioblastoma, most prominently in neural progenitor-like and mesenchymal-like states respectively. These findings implicate these miRNA clusters as potential regulators of glioblastoma intra-tumoral heterogeneity and may serve as valuable biomarkers for cell state identification.

In vivo activity of the second-generation proteasome inhibitor ixazomib against pediatric T-cell acute lymphoblastic leukemia xenografts.

The overall survival rate of patients with T-cell acute lymphoblastic leukemia (T-ALL) is now 90%, although patients with relapsed T-ALL face poor prognosis. The ubiquitin-proteasome system maintains normal protein homeostasis, and aberrations in this pathway are associated with T-ALL. Here we demonstrate the in vitro and in vivo activity of ixazomib, a second-generation orally available, reversible, and selective proteasome inhibitor against pediatric T-ALL cell lines and patient-derived xenografts (PDXs) grown orthotopically in immunodeficient NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJAusb (NSG) mice. Ixazomib was highly potent in vitro, with half-maximal inhibitory concentration (IC50) values in the low nanomolar range. As a monotherapy, ixazomib significantly extended mouse event-free survival of five out of eight T-ALL PDXs in vivo.

In vivo activity of the dual SYK/FLT3 inhibitor TAK-659 against pediatric acute lymphoblastic leukemia xenografts.

BACKGROUND: While children with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, the outcome for certain high-risk pediatric ALL subtypes remains dismal. Spleen tyrosine kinase (SYK) is a prominent cytosolic nonreceptor tyrosine kinase in pediatric B-lineage ALL (B-ALL). Activating mutations or overexpression of Fms-related receptor tyrosine kinase 3 (FLT3) are associated with poor outcome in hematological malignancies. TAK-659 (mivavotinib) is a dual SYK/FLT3 reversible inhibitor, which has been clinically evaluated in several other hematological malignancies. Here, we investigate the in vivo efficacy of TAK-659 against pediatric ALL patient-derived xenografts (PDXs). METHODS: SYK and FLT3 mRNA expression was quantified by RNA-seq. PDX engraftment and drug responses in NSG mice were evaluated by enumerating the proportion of human CD45+ cells (%huCD45+ ) in the peripheral blood. TAK-659 was administered per oral at 60 mg/kg daily for 21 days. Events were defined as %huCD45+ ≥ 25%. In addition, mice were humanely killed to assess leukemia infiltration in the spleen and bone marrow (BM). Drug efficacy was assessed by event-free survival and stringent objective response measures. RESULTS: FLT3 and SYK mRNA expression was significantly higher in B-lineage compared with T-lineage PDXs. TAK-659 was well tolerated and significantly prolonged the time to event in six out of eight PDXs tested. However, only one PDX achieved an objective response. The minimum mean %huCD45+ was significantly reduced in five out of eight PDXs in TAK-659-treated mice compared with vehicle controls. CONCLUSIONS: TAK-659 exhibited low to moderate single-agent in vivo activity against pediatric ALL PDXs representative of diverse subtypes.

In vivo activity of the dual PI3Kδ and PI3Kγ inhibitor duvelisib against pediatric acute lymphoblastic leukemia xenografts.

BACKGROUND: Acute lymphoblastic leukemia (ALL) remains one of the most common causes of cancer-related mortality in children. Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases, and aberrations in the PI3K pathway are associated with several hematological malignancies, including ALL. Duvelisib (Copiktra) is an orally available, small molecule dual inhibitor of PI3Kδ and PI3Kγ, that is Food and Drug Administration (FDA) approved for the treatment of relapsed/refractory chronic lymphocytic leukemia and small lymphocytic lymphoma. Here, we report the efficacy of duvelisib against a panel of pediatric ALL patient-derived xenografts (PDXs). PROCEDURES: Thirty PDXs were selected for a single mouse trial based on PI3Kδ (PIK3CD) and PI3Kγ (PIK3CG) expression and mutational status. PDXs were grown orthotopically in NSG (NOD.Cg-Prkdcscid IL2rgtm1Wjl /SzJAusb) mice, and engraftment was evaluated by enumerating the proportion of human versus mouse CD45+ cells (%huCD45+ ) in the peripheral blood. Treatment commenced when the %huCD45+ reached greater than or equal to 1%, and events were predefined as %huCD45+ greater than or equal to 25% or leukemia-related morbidity. Duvelisib was administered per oral (50 mg/kg, twice daily for 28 days). Drug efficacy was assessed by event-free survival and stringent objective response measures. RESULTS: PI3Kδ and PI3Kγ mRNA expression was significantly higher in B-lineage than T-lineage ALL PDXs (p-values <.0001). Duvelisib was well-tolerated and reduced leukemia cells in the peripheral blood in four PDXs, but with only one objective response. There was no obvious relationship between duvelisib efficacy and PI3Kδ or PI3Kγ expression or mutation status, nor was the in vivo response to duvelisib subtype dependent. CONCLUSIONS: Duvelisib demonstrated limited in vivo activity against ALL PDXs.

Phase II trial of the IDO pathway inhibitor indoximod plus pembrolizumab for the treatment of patients with advanced melanoma.

BACKGROUND: The indoleamine 2,3-dioxygenase (IDO) pathway is a key counter-regulatory mechanism that, in cancer, is exploited by tumors to evade antitumor immunity. Indoximod is a small-molecule IDO pathway inhibitor that reverses the immunosuppressive effects of low tryptophan (Trp) and high kynurenine (Kyn) that result from IDO activity. In this study, indoximod was used in combination with a checkpoint inhibitor (CPI) pembrolizumab for the treatment for advanced melanoma. METHODS: Patients with advanced melanoma were enrolled in a single-arm phase II clinical trial evaluating the addition of indoximod to standard of care CPI approved for melanoma. Investigators administered their choice of CPI including pembrolizumab (P), nivolumab (N), or ipilimumab (I). Indoximod was administered continuously (1200 mg orally two times per day), with concurrent CPI dosed per US Food and Drug Administration (FDA)-approved label. RESULTS: Between July 2014 and July 2017, 131 patients were enrolled. (P) was used more frequently (n=114, 87%) per investigator's choice. The efficacy evaluable population consisted of 89 patients from the phase II cohort with non-ocular melanoma who received indoximod combined with (P).The objective response rate (ORR) for the evaluable population was 51% with confirmed complete response of 20% and disease control rate of 70%. Median progression-free survival was 12.4 months (95% CI 6.4 to 24.9). The ORR for Programmed Death-Ligand 1 (PD-L1)-positive patients was 70% compared with 46% for PD-L1-negative patients. The combination was well tolerated, and side effects were similar to what was expected from single agent (P). CONCLUSION: In this study, the combination of indoximod and (P) was well tolerated and showed antitumor efficacy that is worth further evaluation in selected patients with advanced melanoma.

Measuring Three-Dimensional Temperature Distributions in Steel-Concrete Composite Slabs Subjected to Fire Using Distributed Fiber Optic Sensors.

Detailed information about temperature distribution can be important to understand structural behavior in fire. This study develops a method to image three-dimensional temperature distributions in steel-concrete composite slabs using distributed fiber optic sensors. The feasibility of the method is explored using six 1.2 m × 0.9 m steel-concrete composite slabs instrumented with distributed sensors and thermocouples subjected to fire for over 3 h. Dense point clouds of temperature in the slabs were measured using the distributed sensors. The results show that the distributed sensors operated at material temperatures up to 960 °C with acceptable accuracy for many structural fire applications. The measured non-uniform temperature distributions indicate a spatially distributed thermal response in steel-concrete composite slabs, which can only be adequately captured using approaches that provide a high density of through-depth data points.

Non-Coding RNAs in Pediatric Solid Tumors.

Pediatric solid tumors are a diverse group of extracranial solid tumors representing approximately 40% of childhood cancers. Pediatric solid tumors are believed to arise as a result of disruptions in the developmental process of precursor cells which lead them to accumulate cancerous phenotypes. In contrast to many adult tumors, pediatric tumors typically feature a low number of genetic mutations in protein-coding genes which could explain the emergence of these phenotypes. It is likely that oncogenesis occurs after a failure at many different levels of regulation. Non-coding RNAs (ncRNAs) comprise a group of functional RNA molecules that lack protein coding potential but are essential in the regulation and maintenance of many epigenetic and post-translational mechanisms. Indeed, research has accumulated a large body of evidence implicating many ncRNAs in the regulation of well-established oncogenic networks. In this review we cover a range of extracranial solid tumors which represent some of the rarer and enigmatic childhood cancers known. We focus on two major classes of ncRNAs, microRNAs and long non-coding RNAs, which are likely to play a key role in the development of these cancers and emphasize their functional contributions and molecular interactions during tumor formation.

A Sport-specific Analysis of the Epidemiology of Hip Injuries in National Collegiate Athletic Association Athletes From 2009 to 2014.

PURPOSE: To describe the injury rates, mechanisms, time loss, and rates of surgery for hip/groin injuries in National Collegiate Athletic Association (NCAA) athletes across 25 collegiate sports during the 2009/10 to 2013/14 academic years. METHODS: Data from the 2009/10 to 2013/14 academic years were obtained from the NCAA Injury Surveillance Program (ISP). Rates of hip/groin injuries, mechanism of injury, time lost from competition, and surgical treatment were calculated. Differences between sex-comparable sports were quantified using rate ratios and injury proportion ratios. A sport-specific biomechanical classification system, which included cutting, impingement, overhead/asymmetric, endurance, and flexibility sports, was applied for subgroup analysis. RESULTS: In total, 1,984 hip injuries were reported in 25 NCAA sports, including 9 male and female sports, 3 male-only sports, and 4 female-only sports between the years 2009/10 and 2013/14, resulting in an overall hip injury rate of 53.1/100,000 athletic exposures (AEs). In sex-comparable sports, (basketball, cross-country, lacrosse, ice hockey, indoor track, outdoor track, soccer, swimming, and tennis), men were more commonly affected than women (59.53 vs 42.27 per 100,000 AEs respectively; rate ratio, 1.41; 95% confidence interval, 1.28-1.55). Subgroup analysis demonstrated that the highest rate of hip injuries per 100,000 AEs occurred in impingement sports (96.9). Endurance sports had the highest proportion of injured athletes with time lost >14 days (9.5%). For impingement-type sports, the most common mechanism of injury was no apparent contact (48.2%). The rate of athletes undergoing surgery per 100,000 AEs was highest in impingement-type sports (2.0). CONCLUSIONS: We have identified that impingement-type sports are most frequently associated with hip injuries. Additionally, this study demonstrates that hip injuries sustained in athletes who played impingement-type sports had a significantly higher rate of surgical intervention than other sport classifications. LEVEL OF EVIDENCE: Level III, prognostic study.

Epidemiological Patterns of Patellofemoral Injuries in Collegiate Athletes in the United States From 2009 to 2014.

BACKGROUND: As many as 30% of patients with knee pain seen in sports medicine clinics have complaints related to the patellofemoral joint. There is a paucity of research available regarding patellofemoral injuries, mechanism of injury, and playing time lost in collegiate athletes. PURPOSE: To describe the rates, mechanisms, severity, and potential sex-based differences of patellofemoral injuries in collegiate athletes across 25 National Collegiate Athletic Association (NCAA) sports. STUDY DESIGN: Descriptive epidemiology study. METHODS: Data from the 2009-2010 through the 2013-2014 academic years were obtained from the NCAA Injury Surveillance Program and were analyzed to calculate patellofemoral injury rates, mechanisms of injury, time lost, and need for surgery. Rate ratios and injury proportion ratios were used to quantify discernible differences between sex-comparable sports and timing of injury (ie, practice vs competition), respectively. RESULTS: The overall patellofemoral injury incidence rate was 16.10 per 100,000 athlete-exposures (AEs). Women's volleyball had the highest incidence of all sports (39.57 per 100,000 AEs). Injuries were 66% more likely to occur in competition than during practice. Female athletes experienced significantly more patellofemoral injuries than males in similar sports. Patellar tendinitis accounted for 49.2% of all patellofemoral injuries and was the most common injury in 20 of 25 studied sports. Patellar subluxation accounted for the most total days missed, and patellar dislocation had the highest mean days missed per injury (11.42 days). Patella fracture was the most likely injury to require surgery (80%). CONCLUSION: Patellofemoral injuries were most common in sports that require jumping and quick changes of direction, specifically women's volleyball, men's and women's basketball, and women's soccer. The majority of patellofemoral injuries in this cohort were classified as patellar tendinitis caused by overuse. Most injuries resulted in no competition or practice time lost. This information may contribute to the development of prevention programs aimed at addressing the most prevalent types and mechanisms of injury in each sport to reduce the incidence of patellofemoral injury in these athletes.

Epidemiology of Hip and Groin Injuries in Collegiate Athletes in the United States.

BACKGROUND: Hip and groin pain is a common complaint among athletes. Few studies have examined the epidemiology of hip and groin injuries in collegiate athletes across multiple sports. PURPOSE: To describe the rates, mechanisms, sex-based differences, and severity of hip/groin injuries across 25 collegiate sports. STUDY DESIGN: Descriptive epidemiology study. METHODS: Data from the 2009-2010 through 2013-2014 academic years were obtained from the National Collegiate Athletic Association Injury Surveillance Program (NCAA ISP). The rate of hip/groin injuries, mechanism of injury, time lost from competition, and need for surgery were calculated. Differences between sex-comparable sports were quantified using rate ratios (RRs) and injury proportion ratios (IPRs). RESULTS: In total, 1984 hip/groin injuries were reported, giving an overall injury rate of 53.06 per 100,000 athlete-exposures (AEs). An adductor/groin tear was the most common injury, comprising 24.5% of all injuries. The sports with the highest rates of injuries per 100,000 AEs were men's soccer (110.84), men's ice hockey (104.90), and women's ice hockey (76.88). In sex-comparable sports, men had a higher rate of injuries per 100,000 AEs compared with women (59.53 vs 42.27, respectively; RR, 1.41 [95% CI, 1.28-1.55]). The most common injury mechanisms were noncontact (48.4% of all injuries) and overuse/gradual (20.4%). In sex-comparable sports, men had a greater proportion of injuries due to player contact than women (17.0% vs 3.6%, respectively; IPR, 4.80 [95% CI, 3.10-7.42]), while women had a greater proportion of injuries due to overuse/gradual than men (29.1% vs 16.7%, respectively; IPR, 1.74 [95% CI, 1.46-2.06]). Overall, 39.3% of hip/groin injuries resulted in time lost from competition. Only 1.3% of injuries required surgery. CONCLUSION: Hip/groin injuries are most common in sports that involve kicking or skating and sudden changes in direction and speed. Most hip/groin injuries in collegiate athletes are noncontact and do not result in time lost from competition, and few require surgery. This information can help guide treatment and prevention measures to limit such injuries in male and female collegiate athletes.

Outcomes in the Orthopaedic Sports Medicine Fellowship Match, 2010-2017.

BACKGROUND: Sports medicine is one of the most competitive fellowships in orthopaedic surgery. Despite its popularity, fellowship applicants have limited understanding of the orthopaedic sports medicine fellowship match process. PURPOSE: To define key outcomes in the orthopaedic sports medicine fellowship match, including the overall match rate, number of programs filled, and number of applicants ranked by programs that filled between 2010 and 2017. STUDY DESIGN: Cross-sectional study. METHODS: This study utilized data regarding the orthopaedic sports medicine fellowship match collected by the American Orthopaedic Society for Sports Medicine (AOSSM) from 2010 through 2017. Applicant data included number of applicants, number of matched and unmatched applicants, and percentage of applicants matching into their top choices. Fellowship program data included number of programs participating in the match and number of applicants ranked by filled and unfilled programs. RESULTS: Between 2010 and 2017, the mean number of orthopaedic sports medicine fellowship applicants was 244.8. On average, 92.0% of applicants matched into a fellowship program. The mean number of programs participating in the fellowship match was 92.9, with a mean of 219.9 accredited positions and 5.4 nonaccredited positions. Over the time period studied, a mean of 75.8% of programs matched all available positions. Programs that matched fully ranked 9.0 applicants per position, on average, compared with a mean of 6.5 applicants ranked per position among programs that did not fully match (P = .0016). CONCLUSION: From 2010 to 2017, the number of applicants, positions available, overall match rate, and number of programs participating in the orthopaedic sports medicine fellowship match have remained consistent. The mean number of applicants per position ranked by fully matched fellowship programs was 9.0 compared with a mean of 6.5 applicants per position ranked by programs that did not fully match. These data may be helpful as we look to the future of orthopaedic sports medicine fellowship positions and the match process. In addition, this study reveals characteristics that divide sports medicine fellowship programs that fully match from those that do not. Applicants and/or fellowship program directors may utilize this information to modify their approach to the match process going forward.

Demystifying the "Triple Point: " Technical Nuances of the Fronto-Orbital Advancement.

Removal of the fronto-orbital bandeau is one of the most critical components for procedures designed to correct anomalies of the craniofacial skeleton and remodel the anterior calvarial vault. It is also used to improve exposure of the anterior cranial fossa. It is arguably one of the more difficult portions of some craniofacial procedures. While the technique for fronto-orbito-sphenoid osteotomy has been frequently described, it has only been minimally detailed. Separation of bone in this region remains challenging due to the bone thickness, adjacent vital structures, and limited direct visibility. The present paper describes the anatomy of this particular region, which the authors have termed the "triple point", to facilitate successful osteotomy and avoid potential injury.

Behavior of Steel-Sheathed Shear Walls Subjected to Seismic and Fire Loads.

A series of tests was conducted on six 2.7 m × 3.7 m shear wall specimens consisting of cold-formed steel framing sheathed on one side with sheet steel adhered to gypsum board and on the opposite side with plain gypsum board. The specimens were subjected to various sequences of simulated seismic shear deformation and fire exposure to study the influence of multi-hazard interactions on the lateral load resistance of the walls. The test program was designed to complement a parallel effort at the University of California, San Diego to investigate a six-story building subjected to earthquakes and fires. The test results reported here indicate that the fire exposure caused a shift in the failure mode of the walls from local buckling of the sheet steel in cases without fire exposure, to global buckling of the sheet steel with an accompanying 35 % reduction in lateral load capacity after the wall had been exposed to fire. This behavior appears to be predictable, which is encouraging from the standpoint of residual lateral load capacity under these severe multi-hazard actions.

Altered homeostatic regulation of innate and adaptive immunity in lower gastrointestinal tract GVHD pathogenesis.

Lower gastrointestinal (GI) tract graft-versus-host disease (GVHD) is the predominant cause of morbidity and mortality from GVHD after allogeneic stem cell transplantation. Recent data indicate that lower GI tract GVHD is a complicated process mediated by donor/host antigenic disparities. This process is exacerbated by significant changes to the microbiome, and innate and adaptive immune responses that are critical to the induction of disease, persistence of inflammation, and a lack of response to therapy. Here, we discuss new insights into the biology of lower GI tract GVHD and focus on intrinsic pathways and regulatory mechanisms crucial to normal intestinal function. We then describe multiple instances in which these homeostatic mechanisms are altered by donor T cells or conditioning therapy, resulting in exacerbation of GVHD. We also discuss data suggesting that some of these mechanisms produce biomarkers that could be informative as to the severity of GVHD and its response to therapy. Finally, novel therapies that might restore homeostasis in the GI tract during GVHD are highlighted.

Experimental Analysis of Steel Beams Subjected to Fire Enhanced by Brillouin Scattering-Based Fiber Optic Sensor Data.

This paper presents high temperature measurements using a Brillouin scattering-based fiber optic sensor and the application of the measured temperatures and building code recommended material parameters into enhanced thermomechanical analysis of simply supported steel beams subjected to combined thermal and mechanical loading. The distributed temperature sensor captures detailed, nonuniform temperature distributions that are compared locally with thermocouple measurements with less than 4.7% average difference at 95% confidence level. The simulated strains and deflections are validated using measurements from a second distributed fiber optic (strain) sensor and two linear potentiometers, respectively. The results demonstrate that the temperature-dependent material properties specified in the four investigated building codes lead to strain predictions with less than 13% average error at 95% confidence level and that the Europe building code provided the best predictions. However, the implicit consideration of creep in Europe is insufficient when the beam temperature exceeds 800°C.

Murid herpesvirus-4 exploits dendritic cells to infect B cells.

Dendritic cells (DCs) play a central role in initiating immune responses. Some persistent viruses infect DCs and can disrupt their functions in vitro. However, these viruses remain strongly immunogenic in vivo. Thus what role DC infection plays in the pathogenesis of persistent infections is unclear. Here we show that a persistent, B cell-tropic gamma-herpesvirus, Murid Herpesvirus-4 (MuHV-4), infects DCs early after host entry, before it establishes a substantial infection of B cells. DC-specific virus marking by cre-lox recombination revealed that a significant fraction of the virus latent in B cells had passed through a DC, and a virus attenuated for replication in DCs was impaired in B cell colonization. In vitro MuHV-4 dramatically altered the DC cytoskeleton, suggesting that it manipulates DC migration and shape in order to spread. MuHV-4 therefore uses DCs to colonize B cells.

Interference with dendritic cell populations limits early antigen presentation in chronic γ-herpesvirus-68 infection.

A critical factor influencing the ability of the host to mount a robust immune response against a virus depends on the rapid recruitment of dendritic cells (DCs) presenting Ags. From the outset, this step sets the tempo for subsequent activation of virus-specific T cells. Despite this, how induction of the immune response might be modified by pathogens with the capacity to establish persistence is unclear. In this study, we have characterized the in vivo influence of murine gamma-herpesvirus K3-mediated interference with MHC class I in DCs that drive the initial adaptive immune response. We observed that gamma-herpesvirus could interfere with the very earliest phase of Ag presentation through K3 by directly targeting migratory and lymph node-resident DCs. These results show that a pathogen with the capacity to interfere with early Ag presentation can establish suboptimal conditions for rapid induction of the adaptive immune response and thus favor establishment of viral persistence.

Murid herpesvirus-4 lacking thymidine kinase reveals route-dependent requirements for host colonization.

Gammaherpesviruses infect at least 90 % of the world's population. Infection control is difficult, in part because some fundamental features of host colonization remain unknown, for example whether normal latency establishment requires viral lytic functions. Since human gammaherpesviruses have narrow species tropisms, answering such questions requires animal models. Murid herpesvirus-4 (MuHV-4) provides one of the most tractable. MuHV-4 genomes delivered to the lung or peritoneum persist without lytic replication. However, they fail to disseminate systemically, suggesting that the outcome is inoculation route-dependent. After upper respiratory tract inoculation, MuHV-4 infects mice without involving the lungs or peritoneum. We examined whether host entry by this less invasive route requires the viral thymidine kinase (TK), a gene classically essential for lytic replication in terminally differentiated cells. MuHV-4 TK knockouts delivered to the lung or peritoneum were attenuated but still reached lymphoid tissue. In contrast, TK knockouts delivered to the upper respiratory tract largely failed to establish a detectable infection. Therefore TK, and by implication lytic replication, is required for MuHV-4 to establish a significant infection by a non-invasive route.

In vivo imaging of murid herpesvirus-4 infection.

Luciferase-based imaging allows a global view of microbial pathogenesis. We applied this technique to gammaherpesvirus infection by inserting a luciferase expression cassette into the genome of murine herpesvirus-4 (MuHV-4). The recombinant virus strongly expressed luciferase in lytically infected cells without significant attenuation. We used it to compare different routes of virus inoculation. After intranasal infection of anaesthetized mice, luciferase was expressed in the nose and lungs for 7-10 days and in lymphoid tissue, most consistently the superficial cervical lymph nodes, for up to 30 days. Gastrointestinal infection was not observed. Intraperitoneal infection was very different to intranasal, with strong luciferase expression in the liver, kidneys, intestines, reproductive tract and spleen, but none in the nose or lungs. The nose has not previously been identified as a site of MuHV-4 infection. After intranasal infection of non-anaesthetized mice, it was the only site of non-lymphoid luciferase expression. Nevertheless, lymphoid colonization and persistence were still established, even at low inoculation doses. In contrast, virus delivered orally was very poorly infectious. Inoculation route therefore had a major impact on pathogenesis. Low dose intranasal infection without anaesthesia seems most likely to mimic natural transmission, and may therefore be particularly informative about normal viral gene functions.

An essential role for the proximal but not the distal cytoplasmic tail of glycoprotein M in murid herpesvirus 4 infection.

Murid herpesvirus-4 (MuHV-4) provides a tractable model with which to define common, conserved features of gamma-herpesvirus biology. The multi-membrane spanning glycoprotein M (gM) is one of only 4 glycoproteins that are essential for MuHV-4 lytic replication. gM binds to gN and is thought to function mainly secondary envelopment and virion egress, for which several predicted trafficking motifs in its C-terminal cytoplasmic tail could be important. We tested the contribution of the gM cytoplasmic tail to MuHV-4 lytic replication by making recombinant viruses with varying C-terminal deletions. Removing an acidic cluster and a distal YXXPhi motif altered the capsid distribution somewhat in infected cells but had little effect on virus replication, either in vitro or in vivo. In contrast, removing a proximal YXXPhi motif as well completely prevented productive replication. gM was still expressed, but unlike its longer forms showed only limited colocalization with co-transfected gN, and in the context of whole virus appeared to support gN expression less well. We conclude that some elements of the gM cytoplasmic tail are dispensible for MuHV-4 replication, but the tail as a whole is not.