Hardwiring tissue-specific AAV transduction in mice through engineered receptor expression.

The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.

SETD3 is an actin histidine methyltransferase that prevents primary dystocia.

For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur1. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.

Epizootic of enterocolitis and clostridial overgrowth in NSG and NSG-related mouse strains.

While the immunodeficient status of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) and NSG-related mice provides utility for numerous research models, it also results in increased susceptibility to opportunistic pathogens. Over a 9-week period, a high rate of mortality was reported in a housing room of NSG and NSG-related mice. Diagnostics were performed to determine the underlying etiopathogenesis. Mice submitted for evaluation included those found deceased (n = 2), cage mates of deceased mice with or without diarrhea (n = 17), and moribund mice (n = 8). Grossly, mice exhibited small intestinal and cecal dilation with abundant gas and/or digesta (n = 18), serosal hemorrhage and congestion (n = 6), or were grossly normal (n = 3). Histologically, there was erosive to ulcerative enterocolitis (n = 7) of the distal small and large intestine or widespread individual epithelial cell death with luminal sloughing (n = 13) and varying degrees of submucosal edema and mucosal hyperplasia. Cecal dysbiosis, a reduction in typical filamentous bacteria coupled with overgrowth of bacterial rods, was identified in 18 of 24 (75%) mice. Clostridium spp. and Paeniclostridium sordellii were identified in 13 of 23 (57%) and 7 of 23 (30%) mice, respectively. Clostridium perfringens (7 of 23, 30%) was isolated most frequently. Toxinotyping of C. perfringens positive mice (n = 2) identified C. perfringens type A. Luminal immunoreactivity to several clostridial species was identified within lesioned small intestine by immunohistochemistry. Clinicopathologic findings were thus associated with overgrowth of various clostridial species, though direct causality could not be ascribed. A diet shift preceding the mortality event may have contributed to loss of intestinal homeostasis.

Targeting intramolecular proteinase NS2B/3 cleavages for trans-dominant inhibition of dengue virus.

Many positive-strand RNA viruses translate their genomes as single polyproteins that are processed by host and viral proteinases to generate all viral protein products. Among these is dengue virus, which encodes the serine proteinase NS2B/3 responsible for seven different cleavages in the polyprotein. NS2B/3 has been the subject of many directed screens to find chemical inhibitors, of which the compound ARDP0006 is among the most effective at inhibiting viral growth. We show that at least three cleavages in the dengue polyprotein are exclusively intramolecular. By definition, such a cis-acting defect cannot be rescued in trans This creates the possibility that a drug-susceptible or inhibited proteinase can be genetically dominant, inhibiting the outgrowth of drug-resistant virus via precursor accumulation. Indeed, an NS3-G459L variant that is incapable of cleavage at the internal NS3 junction dominantly inhibited negative-strand RNA synthesis of wild-type virus present in the same cell. This internal NS3 cleavage site is the junction most inhibited by ARDP0006, making it likely that the accumulation of toxic precursors, not inhibition of proteolytic activity per se, explains the antiviral efficacy of this compound in restraining viral growth. We argue that intramolecularly cleaving proteinases are promising drug targets for viruses that encode polyproteins. The most effective inhibitors will specifically target cleavage sites required for processing precursors that exert trans-dominant inhibition.

An Alternate Route for Adeno-associated Virus (AAV) Entry Independent of AAV Receptor.

Determinants and mechanisms of cell attachment and entry steer adeno-associated virus (AAV) in its utility as a gene therapy vector. Thus far, a systematic assessment of how diverse AAV serotypes engage their proteinaceous receptor AAVR (KIAA0319L) to establish transduction has been lacking, despite potential implications for cell and tissue tropism. Here, a large set of human and simian AAVs as well as in silico-reconstructed ancestral AAV capsids were interrogated for AAVR usage. We identified a distinct AAV capsid lineage comprised of AAV4 and AAVrh32.33 that can bind and transduce cells in the absence of AAVR, independent of the multiplicity of infection. Virus overlay assays and rescue experiments in nonpermissive cells demonstrate that these AAVs are unable to bind to or use the AAVR protein for entry. Further evidence for a distinct entry pathway was observed in vivo, as AAVR knockout mice were equally as permissive to transduction by AAVrh32.33 as wild-type mice upon systemic injection. We interestingly observe that some AAV capsids undergo a low level of transduction in the absence of AAVR, both in vitro and in vivo, suggesting that some capsids may have a multimodal entry pathway. In aggregate, our results demonstrate that AAVR usage is conserved among all primate AAVs except for those of the AAV4 lineage, and a non-AAVR pathway may be available to other serotypes. This work furthers our understanding of the entry of AAV, a vector system of broad utility in gene therapy.IMPORTANCE Adeno-associated virus (AAV) is a nonpathogenic virus that is used as a vehicle for gene delivery. Here, we have identified several situations in which transduction is retained in both cell lines and a mouse model in the absence of a previously defined entry receptor, AAVR. Defining the molecular determinants of the infectious pathway of this highly relevant viral vector system can help refine future applications and therapies with this vector.

Inhibition of cellular autophagy deranges dengue virion maturation.

Autophagy is an important component of the innate immune response, directly destroying many intracellular pathogens. However, some pathogens, including several RNA viruses, subvert the autophagy pathway, or components of the pathway, to facilitate their replication. In the present study, the effect of inhibiting autophagy on the growth of dengue virus was tested using a novel inhibitor, spautin-1 (specific and potent autophagy inhibitor 1). Inhibition of autophagy by spautin-1 generated heat-sensitive, noninfectious dengue virus particles, revealing a large effect of components of the autophagy pathway on viral maturation. A smaller effect on viral RNA accumulation was also observed. Conversely, stimulation of autophagy resulted in increased viral titers and pathogenicity in the mouse. We conclude that the presence of functional autophagy components facilitates viral RNA replication and, more importantly, is required for infectious dengue virus production. Pharmacological inhibition of host processes is an attractive antiviral strategy to avoid selection of treatment-resistant variants, and inhibitors of autophagy may prove to be valuable therapeutics against dengue virus infection and pathogenesis.

Variant enterovirus A71 found in immune-suppressed patient binds to heparan sulfate and exhibits neurotropism in B-cell-depleted mice.

Enterovirus A71 (EV-A71) causes hand, foot, and mouth disease outbreaks with neurological complications and deaths. We previously isolated an EV-A71 variant in the stool, cerebrospinal fluid, and blood of an immunocompromised patient who had a leucine-to-arginine substitution on the VP1 capsid protein, resulting in increased heparin sulfate binding. We show here that this mutation increases the virus's pathogenicity in orally infected mice with depleted B cells, which mimics the patient's immune status, and increases susceptibility to neutralizing antibodies. However, a double mutant with even greater heparin sulfate affinity is not pathogenic, suggesting that increased heparin sulfate affinity may trap virions in peripheral tissues and reduce neurovirulence. This research sheds light on the increased pathogenicity of variant with heparin sulfate (HS)-binding ability in individuals with decreased B cell immunity.

The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection.

Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder.

Enterovirus pathogenesis requires the host methyltransferase SETD3.

Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification-mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections.

Helicobacter hepaticus infection promotes colon tumorigenesis in the BALB/c-Rag2(-/-) Apc(Min/+) mouse.

Adenomatous polyposis coli (APC) mutations are linked to human and mouse colorectal cancers. The Apc multiple intestinal neoplasia (Min) mouse mutation causes adenomas to develop throughout the small and large intestines. The BALB-Min (C.B6-Apc(Min/+)) congenic strain was generated by backcrossing into BALB/c the Apc(Min) allele from C57BL/6J-Apc(Min/+) mice. BALB-Min mice have a low tumor multiplicity (27.4 small intestine tumors/mouse) and a relatively long life span (>1 year) that makes them amenable to long-term studies. To investigate the interplay of the adaptive immune system and intestinal tumorigenesis, the immunodeficient compound mutant strain BALB-RagMin (C.Cg-Rag2(-/-) Apc(Min/+)) was generated. BALB-RagMin mice had a significant increase in tumors in the small, but not large, intestine relative to their BALB-Min counterparts (43.0 versus 24.0 tumors/mouse, respectively). The results suggest that the adaptive immune system plays a role in either the elimination or the equilibrium phase of cancer immunoediting in the small intestine in this model. We investigated the effect of the enterohepatic bacterial pathogen Helicobacter hepaticus on liver and intestine tumorigenesis in BALB-RagMin mice. H. hepaticus-infected BALB-RagMin mice developed moderate hepatitis, moderate typhlitis, and mild colitis. There were no differences in small intestine and cecal tumor multiplicity, regionality, or size relative to that in uninfected mice. However, H. hepaticus-infected BALB-RagMin mice had a significant increase in colon tumor incidence relative to uninfected BALB-RagMin mice (23.5% versus 1.7%, respectively). The data suggest that H. hepaticus, which is present in many research colonies, promotes colon tumorigenesis in the BALB-RagMin mouse and that it has the potential to confound colon tumorigenesis studies.

Helicobacter hepaticus promotes azoxymethane-initiated colon tumorigenesis in BALB/c-IL10-deficient mice.

The BALB/c-IL10 null mouse strain develops colitis and colitis-associated adenocarcinomas, and is a model for idiopathic inflammatory bowel disease. We tested the hypotheses that (i) azoxymethane (AOM), a carcinogen that targets the colon, synergizes with the colonic inflammation inherent in the BALB/c-IL10 null mouse resulting in an increase in incidence, multiplicity and/or progression of AOM-induced tumors or colitis-associated adenocarcinomas; and (ii) prior infection with Helicobacter hepaticus, a common enterohepatic bacterial pathogen in many research mouse colonies, increases the incidence, multiplicity and/or progression of AOM-induced colon tumors or colitis-associated adenocarcinomas in the BALB/c-IL10 null mouse. We show that, within the timeframe examined, AOM-induced colon tumors in the BALB/c-IL10 null mouse were grossly and microscopically similar in appearance to AOM-induced colon tumors in the wild type BALB/cJ mouse. No colitis-associated adenocarcinomas were identified. Infection with H. hepaticus prior to AOM-treatment also did not result in colitis-associated adenocarcinomas but did result in a significant increase in the incidence of AOM-induced colon tumors relative to AOM treatment alone. The AOM-induced adenomas were predominantly exophytic and nodular or polypoid and localized to the distal colon. These results suggest that H. hepaticus promotes AOM-induced tumorigenesis in the BALB/c-IL10 null mouse.

Feasibility and biological rationale of repurposing sunitinib and erlotinib for dengue treatment.

There is an urgent need for strategies to combat dengue virus (DENV) infection; a major global threat. We reported that the cellular kinases AAK1 and GAK regulate intracellular trafficking of multiple viruses and that sunitinib and erlotinib, approved anticancer drugs with potent activity against these kinases, protect DENV-infected mice from mortality. Nevertheless, further characterization of the therapeutic potential and underlying mechanism of this approach is required prior to clinical evaluation. Here, we demonstrate that sunitinib/erlotinib combination achieves sustained suppression of systemic infection at approved dose in DENV-infected IFN-α/ß and IFN-γ receptor-deficient mice. Nevertheless, treatment with these blood-brain barrier impermeable drugs delays, yet does not prevent, late-onset paralysis; a common manifestation in this immunodeficient mouse model but not in humans. Sunitinib and erlotinib treatment also demonstrates efficacy in human primary monocyte-derived dendritic cells. Additionally, DENV infection induces expression of AAK1 transcripts, but not GAK, via single-cell transcriptomics, and these kinases are important molecular targets underlying the anti-DENV effect of sunitinib and erlotinib. Lastly, sunitinib/erlotinib combination alters inflammatory cytokine responses in DENV-infected mice. These findings support feasibility of repurposing sunitinib/erlotinib combination as a host-targeted antiviral approach and contribute to understanding its mechanism of antiviral action.

Animal Models of Zika Virus.

Zika virus has garnered great attention over the last several years, as outbreaks of the disease have emerged throughout the Western Hemisphere. Until quite recently Zika virus was considered a fairly benign virus, with limited clinical severity in both people and animals. The size and scope of the outbreak in the Western Hemisphere has allowed for the identification of severe clinical disease that is associated with Zika virus infection, most notably microcephaly among newborns, and an association with Guillian-Barré syndrome in adults. This recent association with severe clinical disease, of which further analysis strongly suggested causation by Zika virus, has resulted in a massive increase in the amount of both basic and applied research of this virus. Both small and large animal models are being used to uncover the pathogenesis of this emerging disease and to develop vaccine and therapeutic strategies. Here we review the animal-model-based Zika virus research that has been performed to date.

Development of Novel ImmunoPET Tracers to Image Human PD-1 Checkpoint Expression on Tumor-Infiltrating Lymphocytes in a Humanized Mouse Model.

PURPOSE: It is well known that cancers exploit immune checkpoints (programmed death 1 receptor (PD-1) and its ligand (PD-L1)) to evade anti-tumor immune responses. Although immune checkpoint (IC) blockade is a promising approach, not all patients respond. Hence, imaging of tumor-infiltrating lymphocytes (TILs) is of high specific interest, as they are known to express PD-1 during activation and subsequent exhaustion in the tumor microenvironment and are thought to be potentially predictive of therapeutic responses to IC blockade. PROCEDURES: We developed immune-tracers for positron emission tomography (PET) to image hPD-1 status of human peripheral blood mononuclear cells (hPBMCs) adoptively transferred to NOD-scid IL-2Rγnull (NSG) mice (hNSG) bearing A375 human skin melanoma tumors. The anti-PD-1 human antibody (IgG; keytruda) was labeled with either Zr-89 or Cu-64 radiometals to image PD-1-expressing human TILs in vivo. RESULTS: [89Zr] Keytruda (groups = 2; NSG-ctl (control) and hNSG-nblk (non-blocking), n = 3-5, 3.2 ± 0.4 MBq/15-16 µg/200 µl) and [64Cu] Keytruda (groups = 3; NSG-ctl, NSG-blk (blocking), and hNSG-nblk; n = 4, 7.4 ± 0.4 MBq /20-25 µg/200 µl) were administered in mice. PET-CT scans were performed over 1-144 h ([89Zr] Keytruda) and 1-48 h ([64Cu] Keytruda) on mice. hNSG mice exhibited a high tracer uptake in the spleen, lymphoid organs and tumors. At 24 h, human TILs homing into melanoma of hNSG-nblk mice exhibited high signal (mean %ID/g ± SD) of 3.8 ± 0.4 ([89Zr] Keytruda), and 6.4 ± 0.7 ([64Cu] Keytruda), which was 1.5- and 3-fold higher uptake compared to NSG-ctl mice (p = 0.01), respectively. Biodistribution measurements of hNSG-nblk mice performed at 144 h ([89Zr] Keytruda) and 48 h ([64Cu] Keytruda) p.i. revealed tumor to muscle ratios as high as 45- and 12-fold, respectively. CONCLUSIONS: Our immunoPET study clearly demonstrates specific imaging of human PD-1-expressing TILs within the tumor and lymphoid tissues. This suggests these anti-human-PD-1 tracers could be clinically translatable to monitor cancer treatment response to IC blockade therapy.

Influence of Genetic Background on Hematologic and Histopathologic Alterations during Acute Granulocytic Anaplasmosis in 129/SvEv and C57BL/6J Mice Lacking Type I and Type II Interferon Signaling.

The role of host type I IFN signaling and its interaction with other immune pathways during bacterial infections is incompletely understood. Type II IFN signaling plays a key role during numerous bacterial infections including granulocytic anaplasmosis (GA) caused by Anaplasma phagocytophilum infection. The function of combined type I and type II IFN signaling and their potential synergism during GA and similar tick-borne diseases is a topic of current research investigation. The goal of this study was to evaluate 2 mouse models of absent type I/type II IFN signaling in experimental A. phagocytophilum infection to determine the effects of background strain. Mice lacking both type I and type II IFN receptor signaling (IFNAR-/-/IFNGR-/-) on either the 129/SvEv or C57BL/6J genetic background were evaluated at days 0, 6, 8, and 12 of infection. Pathogen burden in multiple organs was largely similar between strains of infected mice, with few significant differences. Background strain influenced the immune response to infection. Mice of the 129/SvEv strain developed more severe hematologic abnormalities, particularly more severe leukocytosis with marked neutrophilia and lymphocytosis, throughout acute infection. Histopathologic changes occurred in infected mice of both strains and varied in severity by organ. 129/SvEv mice developed more severe pathologic changes in spleen and bone marrow, whereas C57BL/6J mice developed more severe renal pathology. This work highlights the importance of mouse background strain in dictating pathophysiologic response to infection and informs future work regarding the loss of type I and type II IFN signaling on the immune response during GA.

Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects.

Global health is threatened by emerging viral infections, which largely lack effective vaccines or therapies. Targeting host pathways that are exploited by multiple viruses could offer broad-spectrum solutions. We previously reported that AAK1 and GAK, kinase regulators of the host adaptor proteins AP1 and AP2, are essential for hepatitis C virus (HCV) infection, but the underlying mechanism and relevance to other viruses or in vivo infections remained unknown. Here, we have discovered that AP1 and AP2 cotraffic with HCV particles in live cells. Moreover, we found that multiple viruses, including dengue and Ebola, exploit AAK1 and GAK during entry and infectious virus production. In cultured cells, treatment with sunitinib and erlotinib, approved anticancer drugs that inhibit AAK1 or GAK activity, or with more selective compounds inhibited intracellular trafficking of HCV and multiple unrelated RNA viruses with a high barrier to resistance. In murine models of dengue and Ebola infection, sunitinib/erlotinib combination protected against morbidity and mortality. We validated sunitinib- and erlotinib-mediated inhibition of AAK1 and GAK activity as an important mechanism of antiviral action. Additionally, we revealed potential roles for additional kinase targets. These findings advance our understanding of virus-host interactions and establish a proof of principle for a repurposed, host-targeted approach to combat emerging viruses.

Evaluation of Isoflurane Overdose for Euthanasia of Neonatal Mice.

Neonatal mice (that is, pups younger than 6 d) must be exposed to CO2 for as long as 50 min to achieve euthanasia. Alternatively, other inhalant anesthetic agents have been used to euthanize laboratory rodent species. We investigated the efficacy of isoflurane at saturated vapor pressure to euthanize neonatal mice. Neonatal mice (n = 76; age, 1 or 2 d) were exposed to isoflurane in a sealed, quart-size (0.95-L) plastic bag at room temperature. Righting and withdrawal reflexes were absent in less than 2 min. After 30 min of exposure to isoflurane, pups were removed and monitored for recovery. All pups were cyanotic and showed no detectable signs of life when they were removed from the bag. However, after 30 to 120 min after removal from the bag, 24% of isoflurane-overexposed pups began gasping and then resumed normal respiration and regained a normal pink coloration. These results demonstrate that isoflurane overexposure at saturated vapor pressure for 30 min is insufficient to euthanize neonatal mice and that isoflurane overexposure must be followed by a secondary means of euthanasia.

Proliferative pododermatitis (canker) with intralesional spirochetes in three horses.

Spirochetes were identified histologically in canker (proliferative pododermatitis) in 3 horses. The data suggest that spirochetes may contribute to the pathology of equine chronic foot diseases similar to the manner in which they contribute to the pathology of bovine papillomatous digital dermatitis and contagious ovine digital dermatitis.

Acute paraplegia in a young adult long-evans rat resulting from T-cell lymphoma.

We describe an unusual case of acute paraplegia in a young adult (7.5-month-old) Long-Evans rat that resulted from a spontaneous T-cell lymphoma. At presentation, a neurologic exam revealed normal pelvic limb flexor reflexes, the absence of an anal reflex, and deep pain recognition. Radiographs did not identify any obvious spinal abnormality or osseous trauma, although the liver and spleen were prominent. Hematologic analysis disclosed leukocytosis with atypical lymphocytes. At necropsy, red, friable to gelatinous masses were found associated with the ventral aspect of the vertebral column at the levels of the thoracic and lumbar vertebrae. Impression smears of the mass revealed a monocytic cell population with cells averaging 7 to 10 microm in diameter and having scant cytoplasm and pleomorphic nuclei, characteristics consistent with a lymphoid neoplasm. Histologically, the neoplasm was unencapsulated, poorly demarcated and highly infiltrative, invading and effacing the bone marrow and epidural space of the vertebral column. Neoplastic cells also were identified in the femoral bone marrow, spleen, liver, iliac and sacral lymph nodes, and lung. Immunophenotyping showed the neoplasm to be of T-cell origin. Although the lymphoma did not invade the meninges of the spinal cord, its impingement on the central and peripheral nervous systems resulted in foci of Wallerian degeneration that contributed to the paraplegia. This case report highlights the importance of having lymphoma and leukemia among the differential diagnoses in cases of acute paralysis in rodents.

Suppression of Drug Resistance in Dengue Virus.

UNLABELLED: Dengue virus is a major human pathogen responsible for 400 million infections yearly. As with other RNA viruses, daunting challenges to antiviral design exist due to the high error rates of RNA-dependent RNA synthesis. Indeed, treatment of dengue virus infection with a nucleoside analog resulted in the expected genetic selection of resistant viruses in tissue culture and in mice. However, when the function of the oligomeric core protein was inhibited, no detectable selection of drug resistance in tissue culture or in mice was detected, despite the presence of drug-resistant variants in the population. Suppressed selection of drug-resistant virus correlated with cooligomerization of the targeted drug-susceptible and drug-resistant core proteins. The concept of "dominant drug targets," in which inhibition of oligomeric viral assemblages leads to the formation of drug-susceptible chimeras, can therefore be used to prevent the outgrowth of drug resistance during dengue virus infection. IMPORTANCE: Drug resistance is a major hurdle in the development of effective antivirals, especially those directed at RNA viruses. We have found that one can use the concept of the genetic dominance of defective subunits to "turn cousins into enemies," i.e., to thwart the outgrowth of drug-resistant viral genomes as soon as they are generated. This requires deliberate targeting of larger assemblages, which would otherwise rarely be considered by antiviral researchers.