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Novel urinary protein biomarkers have recently been identified and qualified in rats for the early detection of renal injury in drug development studies. However, there are few reports on the utility of these renal biomarkers in mice, another important and widely used preclinical animal species for drug development studies. The purpose of this study was to assess the value of these recently qualified biomarkers for the early detection of drug-induced kidney injury (DIKI) in different strains of mice using multiple assay panels. To this end, we evaluated biomarker response to kidney injury induced by several nephrotoxic agents including amphotericin B, compound X, and compound Y. Several of the biomarkers were shown to be sensitive to DIKI in mice. When measured, urinary albumin and neutrophil gelatinase-associated lipocalin were highly sensitive to renal tubular injury, regardless of the assay platforms, mouse strain, and nephrotoxic agents. Depending on the type of renal tubular injury, kidney injury molecule-1 was also highly sensitive, regardless of the assay platforms and mouse strain. Osteopontin and cystatin C were modestly to highly sensitive to renal tubular injury, but the assay type and/or the mouse strain should be considered before using these biomarkers. Calbindin D28 was highly sensitive to injury to the distal nephron in mice. To our knowledge, this is the first report that demonstrates the utility of novel urinary biomarkers evaluated across multiple assay platforms and nephrotoxicants in different mice strains with DIKI. These results will help drug developers make informed decisions when selecting urinary biomarkers for monitoring DIKI in mice for toxicology studies.

Transforming growth factor β (TGFβ) signaling has been recently shown to reduce antitumor response to PD-L1 blockade, leading to a renewed enthusiasm in developing anti-TGFβ therapies for potential combination with cancer immunotherapy agents. Inhibition of TGFβ signaling in nonclinical toxicology species is associated with serious adverse toxicities including cardiac valvulopathies and anemia. Previously, cardiovascular toxicities have been thought to be limited to small molecule inhibitors of TGFβ receptor and not considered to be a liability associated with pan-TGFβ neutralizing monoclonal antibodies (mAbs). Here, we report the toxicity findings associated with a potent pan-TGFβ neutralizing mAb (pan-TGFβ mAb; neutralizes TGFβ1, 2, and 3) after 5 weekly intravenous doses of 10, 30, and 100 mg/kg, followed by a 4-week recovery period, in mice and cynomolgus monkeys. Mortality was observed due to acute bleeding and cardiovascular toxicity in mice at ≥ 30 mg/kg and prolonged menstruation in female monkeys at 100 mg/kg. Additional findings considered to be on-target exaggerated pharmacology included generalized bleeding and cardiovascular toxicity in mice and monkeys; histopathologic changes in the teeth, tongue, and skin in mice; and abnormal wound healing and microscopic pathology in the bone in monkeys. Importantly, our data indicate that the cardiovascular toxicities associated with the inhibition of TGFβ signaling are not limited to small molecule inhibitors but are also observed following administration of a potent pan-TGFβ inhibiting mAb.

Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase‐dependent apoptosis and caspase‐independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild‐type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections., Inhibiting RIP1 has benefits to skin inflammation but not certain viral infections.

The interpretation of clinical pathology results from nonclinical safety studies is a fundamental component in hazard identification of new drug candidates. The ever-increasing complexity of nonclinical safety studies and sophistication of modern analytical methods have made the interpretation of clinical pathology information by a highly trained subject matter expert imperative. Certain interpretive techniques are particularly effective in the identification and characterization of clinical pathology effects. The purpose of this manuscript is to provide an overview of contemporary interpretive practices for clinical pathology results and to provide nonbinding recommendations aimed at improving consistency, quality, and overall value of clinical pathology interpretations generated in support of nonclinical toxicology studies.

Clinical pathology reporting practices are diverse among individuals and organizations involved in nonclinical toxicology studies. Clear, informative, and consistent reporting of clinical pathology results increases their value and avoids misinterpretation, resulting in decreased drug development costs. In recent years, certain common practices in clinical pathology reporting have been embraced by industry leaders and more consistently utilized across the pharmaceutical industry. The purpose of this manuscript is to review current clinical pathology reporting practices and to provide nonbinding suggestions to improve consistency, quality, and value of clinical pathology reports generated in support of nonclinical toxicology studies.

Novel urinary protein biomarkers have recently been identified and qualified in rats for the early detection of renal injury in drug development studies. However, there seems to be no standardized normalization method for analyzing these urinary biomarkers, as some users normalize with urinary creatinine (uCr), urine volume (uVol), or leave biomarker un-normalized. More recently, urinary cystatin C is also emerging as a urinary biomarker normalizer, given some of its characteristics as a glomerular filtration marker. The purpose of this study was to identify an optimal drug-induced kidney injury biomarker normalization method that can be adopted more uniformly in the field. To this end, we compared the variability of uVol, urinary cystatin C, and Cr in healthy rats; we evaluated the sensitivity of the renal biomarkers to renal injury after normalization with uVol, uCr, and cystatin C in rats with cisplatin-induced renal injury. We showed that, over time, uCr was less variable than urinary cystatin C and uVol. When the renal biomarkers were normalized with the 3 normalizing end points, the biomarkers showed (1) least variability following normalization with Cr in healthy animals and (2) poor sensitivity when normalized with urinary cystatin C in animals with renal injury. Overall, the results suggested that uCr is better than urinary cystatin C and uVol for normalizing renal biomarkers in rats under controlled preclinical conditions. To our knowledge, this is the first report that compared the variability of uVol, cystatin C, and Cr in the context of renal biomarkers’ normalization.

We report the unique pathogenesis and presentation of a rapidly progressive B-cell lymphoma in a 3-year-old female cynomolgus monkey on day 50 of a 13-week toxicity study. Clinical pathology evaluation revealed a marked leukocytosis with bicytopenia. A serum protein electrophoresis was consistent with monoclonal gammopathy. The architecture of the lymph node, spleen, and thymus were variably effaced by neoplastic cells, which also infiltrated other tissues. Immunohistochemistry of the affected tissues confirmed a predominant population of CD20+, CD79a+, CD3-, CD68-, and CD34-neoplastic cells. The full data best support a diagnosis of Stage V lymphoma. Nextgen sequencing and negative prestudy serology results suggested a recent infection by macaque lymphocryptovirus (mLCV) with a unique transcriptional profile comparable with a rarely observed direct LCV infection model. This infection model might be associated with a temporary lack of an LCV antigen-specific cytotoxic T-cell adaptive immune response. Consistent with the established mechanisms of LCV-related lymphoproliferation, MYC and BCL2L11 gene expression were increased and decreased, respectively. While there was no overt immunosuppression, immunophenotyping revealed the index animal had a relatively low NK cell count, which further decreased by >50% on day 24 of the study. In addition to the temporary lack of adaptive immunity, the low NK cell counts were suggestive of an impaired innate immunity to control the virally-transformed cells and the subsequent unchecked lymphoproliferation. To our knowledge, this is the first report of a Stage V lymphoma with a unique pathogenesis in an otherwise immunocompetent cynomolgus monkey.

CASE DESCRIPTION A 4-year-old sexually intact male pet guinea pig (Cavia porcellus) was evaluated for a routine wellness examination. CLINICAL FINDINGS During physical examination, a small mass was palpated in the cranial aspect of the abdomen. Abdominal radiographic and ultrasonographic findings were suggestive of a gastric mass. Cytologic evaluation of a fine-needle aspirate of the mass was indicative of spindle cell proliferation most consistent with a sarcoma. TREATMENT AND OUTCOME The patient was anesthetized, and an exploratory laparotomy and partial gastrectomy were performed to resect the gastric mass. Histologic and immunohistochemical examinations of the mass revealed that it was a gastric leiomyoma. The patient recovered from surgery without complications. No evidence of mass recurrence was observed during an abdominal ultrasonographic examination performed approximately 19 months after surgery. CLINICAL RELEVANCE To our knowledge, this was the first report of the clinical diagnosis and successful surgical treatment of a gastric neoplasm in a guinea pig. Gastric leiomyomas are not uncommon in guinea pigs, and although benign, they can cause clinical signs if they become large enough to impair gastric function. Gastrointestinal surgery should be considered as a treatment option for guinea pigs with similar gastric neoplasms.

RO7297089 is a bispecific antibody (IgG-scFv) targeting Bcell maturation antigen (BCMA) and CD16a (FcγRIIIA) that is being developed for the treatment of multiple myeloma (MM). BCMA is exclusively expressed on plasmablasts and differentiated plasma cells (PCs), and is overexpressed on malignant PCs in MM patients. CD16a is expressed on natural killer (NK) cells, monocytes, mast cells, and macrophages. Herein, we characterized the mode of action and safety profile of RO7297089 in vitro and in vivo. RO7297089 showed potent cell killing when using BCMA+ MM tumor cell lines as target cells and human peripheral blood mononuclear cells, NK cells or macrophages as effector cells. Minimal increases in TNFα (2x) and IFNγ (4x), but not other cytokines/chemokines, were observed compared to the vehicle control treatment only in the presence of the BCMA+ MM tumor cell line up to the concentration tested. This suggests that, unlike T-cell engagers, the risk of cytokine release syndrome in patients receiving RO7297089 is low. Cynomolgus monkey is the only relevant nonclinical species for RO7297089 as it showed binding to both recombinant CD16 and BCMA with comparable affinity to human antigens. Following five weekly intravenous administrations to monkeys at 0, 15, and 50 mg/kg, RO7297089 was well tolerated. In line with the mechanism of action, there were no test article-related cytokine increases or adverse findings observed in both dose levels. Systemic exposure of RO7297089 was approximately dose proportional from 15 to 50 mg/kg. Anti-drug antibodies (ADA) were observed in some animals at both dose levels, and ADA-related decreases in concentrations were observed at only 15 mg/kg. To evaluate in vivo activity, RO7297089-related effects on total plasma sBCMA and PCs were assessed. Elevations of sBCMA levels (100x) were observed post dose at both dose levels, and these effects returned to predose levels in animals that did not maintain concentrations at 15 mg/kg, suggesting that RO7297089 bound to and stabilized circulating cynomolgus sBCMA. Time- and dose-dependent reductions in serum IgM levels were observed at both dose levels. Changes in PC numbers were not detected by immunophenotyping; however, gene expression analysis of PC markers was included and demonstrated clear reductions in mRNA expression levels of PC markers including BCMA and J-chain in blood at both dose levels, suggesting reductions in BCMA+ cells. Collectively, these studies suggest that RO7297089 selectively kills BCMA+ cells by engaging CD16a-positive immune cells and has a favorable safety profile. Citation Format: Satoko Kakiuchi-Kiyota, Melissa M. Schutten, Adeyemi O. Adedeji, Hao Cai, Robert Hendricks, Luna Liu, Sivan Cohen, Aaron M. Fullerton, Nicholas Corr, Lanlan Yu, Denise de Almeida Nagata, Shelly Zhong, Michael Dillon, Christoph Spiess, Steve R. Leong, Bing Zheng, Susanne Wingert, Uwe Reusch, Stefan Knackmuss, Thorsten Ross, Andrew Polson, Ayse M. Ovacik. Preclinical pharmacology and safety of RO7297089, a novel anti-BCMA/CD16a bispecific antibody for the treatment of multiple myeloma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4556.

Background Follicular tumors and cysts are common skin lesions in dogs. Both are distinguished based on their cellular origin (matrical, isthmus, or infundibular cells) and the type of keratin they produce. Typically, differentiation requires histopathology, as all these lesions often have similar cytologic features. Objectives The goal of this retrospective study was to identify unique cytologic features that may assist differentiation of canine benign follicular tumors and cysts at cytology, using histopathology as gold standard. Methods Electronic medical records of the University of California-Davis Veterinary Medical Teaching Hospital were searched for diagnoses of follicular tumors and cysts in dogs that had both histopathologic and cytologic diagnoses between January 2000 and December 2013. Cytologic specimens were reassessed in a blinded manner for the presence and type of background, cells, noncellular elements, and inflammation. Results Forty-six samples were included in the study. Follicular cysts (n = 25) and infundibular keratinizing acanthomas (n = 5) contained sheets of keratinized anucleate to nucleated squamous cells. Trichoepitheliomas (n = 8) had 2 primary cytologic presentations: those with primarily basaloid cells that appeared to be undergoing keratinization (3/8; 38%) and those with keratinizing anucleate to nucleated squamous cells (5/8; 63%). Trichoblastomas (n = 8) had pink matrix with spindle cells and contained cohesive clusters of basaloid cells. Conclusion Trichoblastomas, and to a lesser extent, trichoepitheliomas, have unique cytologic features with the potential to distinguish them from other benign follicular tumors and cysts. These results are an important first step toward improving the diagnostic specificity of cytologic examination of cutaneous follicular lesions.

Coronaviruses are positive stranded RNA viruses that cause respiratory, enteric and central nervous system diseases in many species, including humans. Until recently, the relatively low burden of disease in humans caused by few of these viruses impeded the development of coronavirus specific therapeutics. However, the emergence of severe acute respiratory syndrome coronavirus (SARS-CoV), and more recently, Middle East respiratory syndrome coronavirus (MERS-CoV), has impelled the development of such drugs. This review focuses on some newly identified SARS-CoV inhibitors, with known mechanisms of action and their potential to inhibit the novel MERS-CoV. The clinical development of optimized versions of such compounds could be beneficial for the treatment and control of SARS-CoV, the current MERS-CoV and other future SARS-like epidemics.

We have previously shown that SSYA10-001 blocks severe acute respiratory syndrome coronavirus (SARS-CoV) replication by inhibiting SARS-CoV helicase (nsp13). Here, we show that SSYA10-001 also inhibits replication of two other coronaviruses, mouse hepatitis virus (MHV) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV). A putative binding pocket for SSYA10-001 was identified and shown to be similar in SARS-CoV, MERS-CoV, and MHV helicases. These studies show that it is possible to target multiple coronaviruses through broad-spectrum inhibitors.

Coronaviruses are known to cause a wide range of diseases in humans and animals. Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel coronavirus discovered in 2012 and is responsible for acute respiratory syndrome in humans in the Middle East, Europe, North Africa, and the United States of America. Helicases are motor proteins that catalyze the processive separation of double-stranded nucleic acids into two single-stranded nucleic acids by utilizing the energy derived from ATP hydrolysis. MERS-CoV helicase is one of the most important viral replication enzymes of this coronavirus. Herein, we report the first bacterial expression, enzyme purification, and biochemical characterization of MERS-CoV helicase. The knowledge obtained from this study might be used to identify an inhibitor of MERS-CoV replication, and the helicase might be used as a therapeutic target., Middle East respiratory syndrome coronavirus (MERS-CoV) helicase is a superfamily 1 helicase containing seven conserved motifs. We have cloned, expressed, and purified a Strep-fused recombinant MERS-CoV nonstructural protein 13 (M-nsp13) helicase. Characterization of its biochemical properties showed that it unwound DNA and RNA similarly to severe acute respiratory syndrome CoV nsp13 (S-nsp13) helicase. We showed that M-nsp13 unwound in a 5′-to-3′ direction and efficiently unwound the partially duplex RNA substrates with a long loading strand relative to those of the RNA substrates with a short or no loading strand. Moreover, the Km of ATP for M-nsp13 is inversely proportional to the length of the 5′ loading strand of the partially duplex RNA substrates. Finally, we also showed that the rate of unwinding (ku) of M-nsp13 is directly proportional to the length of the 5′ loading strand of the partially duplex RNA substrate. These results provide insights that enhance our understanding of the biochemical properties of M-nsp13. IMPORTANCE Coronaviruses are known to cause a wide range of diseases in humans and animals. Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel coronavirus discovered in 2012 and is responsible for acute respiratory syndrome in humans in the Middle East, Europe, North Africa, and the United States of America. Helicases are motor proteins that catalyze the processive separation of double-stranded nucleic acids into two single-stranded nucleic acids by utilizing the energy derived from ATP hydrolysis. MERS-CoV helicase is one of the most important viral replication enzymes of this coronavirus. Herein, we report the first bacterial expression, enzyme purification, and biochemical characterization of MERS-CoV helicase. The knowledge obtained from this study might be used to identify an inhibitor of MERS-CoV replication, and the helicase might be used as a therapeutic target.

We report key mechanistic differences between the reverse transcriptases (RT) of human immunodeficiency virus type-1 (HIV-1) and of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus that can infect human cells. Steady and pre-steady state kinetics demonstrated that XMRV RT is significantly less efficient in DNA synthesis and in unblocking chain-terminated primers. Surface plasmon resonance experiments showed that the gammaretroviral enzyme has a remarkably higher dissociation rate (k(off)) from DNA, which also results in lower processivity than HIV-1 RT. Transient kinetics of mismatch incorporation revealed that XMRV RT has higher fidelity than HIV-1 RT. We identified RNA aptamers that potently inhibit XMRV, but not HIV-1 RT. XMRV RT is highly susceptible to some nucleoside RT inhibitors, including Translocation Deficient RT inhibitors, but not to non-nucleoside RT inhibitors. We demonstrated that XMRV RT mutants K103R and Q190M, which are equivalent to HIV-1 mutants that are resistant to tenofovir (K65R) and AZT (Q151M), are also resistant to the respective drugs, suggesting that XMRV can acquire resistance to these compounds through the decreased incorporation mechanism reported in HIV-1.

More than a year ago, a novel coronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV), was first identified as the causative agent of a severe pneumonia in several patients in the Middle East [1]. As of August 5, 2013, a total of 94 cases have been identified in the Middle East (Saudi Arabia, Jordan, Qatar, and the United Arab Emirates), Europe (United Kingdom, France, Italy, and Germany), and North Africa (Tunisia), with a case fatality rate of 56%. Since this coronavirus is closely related to severe acute respiratory syndrome coronavirus (SARS-CoV), an epidemic that was short-lived but alarming in 2002–2003 that resulted in approximately 8000 cases and 800 deaths, several health agencies that include governmental, public health, clinical, and laboratory authorities have all moved rapidly to respond to this new virus outbreak. Following identification of MERS-CoV, details about its genomic sequence and organization, species tropism, and host-cell receptor (dipeptidyl peptidase 4) have been reported [2,3]. Although there have been several reports of inhibitors of SARS-CoV, to date, there are no approved drugs or vaccines for the treatment of SARS-CoV. Studies have shown that ribavirin has a very minimal effect in controlling adverse outcomes from SARS-CoV infection. Its use has attracted a lot of criticism due to its unproven and low efficacy and unwarranted side effects [4]. At non-toxic concentrations, ribavirin has no direct in vitro activity against SARS-CoV [5]. Among some of the promising agents for the control of SARS-CoV infection are interferons. A large number of recombinant interferons that belong to the three classes (α, β and γ) have been tested for their antiviral activities against SARS-CoV in vitro. Interferon-α and -β were shown to be active against SARS-CoV, with interferon-β being the most active of the three [6]. More recently, Falzarano et al [7] proposed a potential therapeutic regimen for the treatment of MERS-CoV based on cell-based experiments. In this report, the authors compared the use of both interferon-α2b and ribavirin individually or in combination for the inhibition of MERS-CoV in two different cell lines, Vero and LLC-MK2. When used individually, to completely eliminate the cytopathic effect (CPE) of SARS-CoV infection, each drug required concentrations as high as 1000 U/ml interferon-α2b (IFN- α2b) and 200 µg/ml ribavirin. However, when the two drugs were combined they acted synergistically, and only 125 U/ml IFN-α2b and 25 µg/ml ribavirin were required to completely eliminate the CPE. This represented an 8- and 16-fold decrease in the amount of IFN-α2b and ribavirin respectively, which is required to attain the same reduction when used individually. While the combination therapy portends to be a promising strategy, these concentrations are still very much higher than the current doses at which these two drugs are being used for Hepatitis C virus (HCV) infections in humans [8]. High doses of ribavirin have been associated with hemolytic anemia, elevated transaminase levels and bradycardia [9]. Despite the lower doses of these drugs for the treatment of HCV, many side effects accompany this combination therapy, which include depression, suicide, relapse of drug abuse/overdose, bacterial infections and many others (http://www.hepatitis.va.gov/provider/reviews/treatment-side-effects.asp#S1X). Nonetheless, given the severity of this disease and the lack of any available treatments, this is a promising finding that should be further explored. Given the similarity of MERS-CoV with SARS-CoV, what lessons can we learn from past work on SARS-CoV, or other positive strand RNA viruses? MERS-CoV was recently shown to use dipeptidyl peptidase 4 as its functional receptor to gain entry into cells [3]. While this virus is utilizing a receptor different than the one used by SARS and other respiratory coronaviruses, there appears to be some similarities in their mechanisms of entry. MERS-CoV has been shown to require cathepsins B and L for entry, similar to SARS-CoV [10]. Cathepsin L inhibitors have been shown to prevent SARS-CoV entry in SARS-CoV-pseudotyped assays (SARS-CoV env / HIV core) [11]. Published cathepsin L inhibitors include oxocarbazate, MDL28170 and SSAA09E1. MDL28170 and oxocarbazate inhibit SARS-CoV-pseudotyped virus with EC50s of ~100 nM and ~300 nM respectively [12,13]. These two compounds appear to be peptidomimetic in nature, thereby limiting their use only to parenteral administration if they were to be considered for therapeutic purposes. SSAA09E1, a non-peptidomimetic small molecule that inhibited SARS-CoV-pseudotyped virus with an EC50 of ~7 µM can also inhibit live SARS-CoV in Vero cells (unpublished data). Therefore, any of these cathepsin L inhibitors would be worth testing for inhibition of MERS-CoV as well as of other coronaviruses that require cathepsin L for entry. In thinking about treatments of viral infections, one has to also consider the challenge of drug resistance, which is the result of the ability of viruses to rapidly evolve and escape treatments through multiple and diverse mechanisms of resistance. There is an extensive body of clinical data on development of resistance against antivirals that are used in the clinic for the treatment of Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), and Hepatitis C Virus (HCV) infections. For HIV, remarkable progress has been made with highly active anti-retroviral therapies (HAART), which are based on combinations of drugs that almost exclusively target directly the virus. HAART regimens significantly suppress the viral load in the blood plasma of HIV patients, resulting in significant decreases in HIV-related deaths. However, prolonged treatment inevitably results in resistance to all of these available drugs. [14–16]. Similar benefits have been reported in the treatment of HBV-infected patients with antiretrovirals. For many years, HCV has been treated with a combination therapy of pegylated interferon and ribavirin. Clinical trials have shown sustained virological response rates (SVRs) of ~50% (efficacy), which does not even provide equivalent results in clinical practice [17]. HCV has also evolved various mechanisms to develop resistance to these therapies. For example, HCV circumvents the IFN response by blocking downstream signaling actions via STAT1, STAT2, IRF9 and JAK-STAT pathways [18,19]. Hence, MERS coronavirus can also develop resistance to IFN treatment. Ribavirin is a nucleoside analog that after activation by host kinases to a nucleotide, it can be used by viral polymerases and incorporated into the nascent viral genomes, causing mutations by allowing base mismatches. During the amplification of hepatitis C replicons, error frequency is known to increase upon ribavirin treatment. Hence, ribavirin's antiviral activity is likely to result from error catastrophe caused by increased mutagenesis of viral genomes thereby reducing the fitness of the virus. Another mechanism through which ribavirin may exhibit its antiviral activity is by depletion of intracellular GTP pools (by inhibition of the cellular Inosine monophosphate dehydrogenase [IMPDH] by the 5′-monophosphate metabolite of ribavirin) [20]. This mechanism may be useful in explaining the drug's general cytotoxic and anti-DNA replication effect as well as some effect on DNA viral replication. While reports of viral resistance to ribavirin are rare, its reliance on host factors to be able to convert to its mono- and tri-phosphate active forms stands as a potential problem. Darryl Falzarano et al [7] showed the difference in the efficacies of ribavirin depending on the cell types, as ribavirin was more effective in LLC-MK2 cells than the Vero cells. Since, MERS-CoV naturally infects respiratory epithelial cell, it will be more expedient to monitor the effect of ribavirin in respiratory epithelial cells to see if comparable viral inhibition will be observed. With limited efficacy of this therapy, coupled with multiple side effects and resistance development, many scientists have been working on IFN-free therapy that involves development and the use of small molecule compounds that directly inhibit the viral replication proteins. The efficacies of these compounds are being tested alone or in combination with ribavirin or pegylated IFNs. These compounds are referred to as “Direct Acting Antivirals”. Examples include the recently approved boceprevir and telaprevir NS3/4A protease inhibitors, as well as NS5A inhibitors and NS5B polymerase inhibitors [21] that are in clinical trials. For coronaviruses, several reports of SARS-CoV small molecule compounds that inhibit the viral replication proteins, or prevent entry of the virus into cells, are well documented [11,12,22,23]. All these compounds, with known mechanisms of action, introduce potential leads for the development of MERS and other coronaviruses therapeutics. In conclusion, the recent report that interferon-α2b and ribavirin have some synergistic activity in cell-based assays is indeed encouraging, as these drugs are currently used to treat hepatitis C and are therefore widely available even though they are imperfect but with known pharmacological properties. Moreover, the study highlights how past research in related viruses can provide useful insights in the treatment of emerging diseases.

Severe acute respiratory syndrome (SARS) is an infectious and highly contagious disease that is caused by SARS coronavirus (SARS-CoV) and for which there are currently no approved treatments. We report the discovery and characterization of small-molecule inhibitors of SARS-CoV replication that block viral entry by three different mechanisms. The compounds were discovered by screening a chemical library of compounds for blocking of entry of HIV-1 pseudotyped with SARS-CoV surface glycoprotein S (SARS-S) but not that of HIV-1 pseudotyped with vesicular stomatitis virus surface glycoprotein G (VSV-G). Studies on their mechanisms of action revealed that the compounds act by three distinct mechanisms: (i) SSAA09E2 { N -[[4-(4-methylpiperazin-1-yl)phenyl]methyl]-1,2-oxazole-5-carboxamide} acts through a novel mechanism of action, by blocking early interactions of SARS-S with the receptor for SARS-CoV, angiotensin converting enzyme 2 (ACE2); (ii) SSAA09E1 {[( Z )-1-thiophen-2-ylethylideneamino]thiourea} acts later, by blocking cathepsin L, a host protease required for processing of SARS-S during viral entry; and (iii) SSAA09E3 [ N -(9,10-dioxo-9,10-dihydroanthracen-2-yl)benzamide] also acts later and does not affect interactions of SARS-S with ACE2 or the enzymatic functions of cathepsin L but prevents fusion of the viral membrane with the host cellular membrane. Our work demonstrates that there are at least three independent strategies for blocking SARS-CoV entry, validates these mechanisms of inhibition, and introduces promising leads for the development of SARS therapeutics.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus that infects both goats and sheep and is closely related to maedi-visna virus that infects sheep; collectively, these viruses are known as small ruminant lentiviruses (SRLV). Infection of goats and sheep with SRLV typically results in discrete inflammatory diseases which include arthritis, mastitis, pneumonia or encephalomyelitis. SRLV-infected animals concurrently demonstrating lentivirus-associated lesions in tissues of lung, mammary gland, joint synovium and the central nervous system are either very rare or have not been reported. Here we describe a novel CAEV promoter isolated from a sheep with multisystemic lentivirus-associated inflammatory disease including interstitial pneumonia, mastitis, polyarthritis and leukomyelitis. A single, novel SRLV promoter was cloned and sequenced from five different anatomical locations (brain stem, spinal cord, lung, mammary gland and carpal joint synovium), all of which demonstrated lesions characteristic of lentivirus associated inflammation. This SRLV promoter isolate was found to be closely related to CAEV promoters isolated from goats in northern California and other parts of the world. The promoter was denoted CAEV-ovine-MS (multisystemic disease); the stability of the transcription factor binding sites within the U3 promoter sequence are discussed.

The non-structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a helicase that separates double-stranded RNA (dsRNA) or DNA (dsDNA) with a 5' → 3' polarity, using the energy of nucleotide hydrolysis. We determined the minimal mechanism of helicase function by nsp13. We showed a clear unwinding lag with increasing length of the double-stranded region of the nucleic acid, suggesting the presence of intermediates in the unwinding process. To elucidate the nature of the intermediates we carried out transient kinetic analysis of the nsp13 helicase activity. We demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base-pairs (bp) each, with a catalytic rate of 30 steps per second. Therefore the net unwinding rate is ~280 base-pairs per second. We also showed that nsp12, the SARS-CoV RNA-dependent RNA polymerase (RdRp), enhances (2-fold) the catalytic efficiency of nsp13 by increasing the step size of nucleic acid (RNA/RNA or DNA/DNA) unwinding. This effect is specific for SARS-CoV nsp12, as no change in nsp13 activity was observed when foot-and-mouth-disease virus RdRp was used in place of nsp12. Our data provide experimental evidence that nsp13 and nsp12 can function in a concerted manner to improve the efficiency of viral replication and enhance our understanding of nsp13 function during SARS-CoV RNA synthesis.