Human Rhinovirus Type Research Articles

Patient survival on the dose escalation phase of the Oncolytic Polio/Rhinovirus Recombinant (PVSRIPO) against WHO grade IV malignant glioma (MG) clinical trial compared to historical controls.

2061Background: The live attenuated oral (SABIN) serotype 1 poliovirus vaccine was modified to contain a heterologous internal ribosomal entry site stemming from human rhinovirus type 2, creating PVSRIPO. PVSRIPO recognizes CD155, an oncofetal cell adhesion molecule and tumor antigen widely expressed ectopically in malignancy. We report the survival results of the dose-finding portion evaluating PVSRIPO delivered intratumorally by convection-enhanced delivery (CED) compared to a historical control group of patients (pts) treated at Duke. Methods: Eligible pts on trial were adults with recurrent supratentorial WHO grade IV MG; solitary tumor 1-5cm in diameter; ≥ 4 weeks after chemotherapy, bevacizumab or study drug; adequate organ function; KPS ≥ 70%; and positive anti-polio titer. A historical group of adult recurrent WHO grade IV mg pts treated at Duke between 1/01/07-12/15/14 was retrospectively identified based on presence of a solitary tumor 1-5cm in diameter, KPS ≥ 70%, and absence of clinical declin...

Oncolytic polio/rhinovirus recombinant (PVSRIPO) against recurrent glioblastoma (GBM): Optimal dose determination.

2068 Background: The live attenuated oral (SABIN) serotype 1 poliovirus vaccine was modified to contain a heterologous internal ribosomal entry site stemming from human rhinovirus type 2, creating ...

Subclinical infection and dosing in primary immunodeficiencies.

Subclinical infection and dosing in primary immunodeficiencies.

AT-21 * FINAL RESULTS OF A PHASE 1 TRIAL OF AN ONCOLYTIC POLIO/RHINOVIRUS RECOMBINANT (PVSRIPO) AGAINST RECURRENT GLIOBLASTOMA (GBM)

BACKGROUND: PVSRIPO is the live attenuated, oral (SABIN) serotype 1 poliovirus vaccine containing a heterologous internal ribosomal entry site stemming from human rhinovirus type 2. PVSRIPO recognizes nectin-like molecule-5, an oncofetal cell adhesion molecule and tumor antigen widely expressed ectopically in malignancy. We report results of a phase 1 trial evaluating PVSRIPO delivered intratumorally by convection-enhanced delivery (CED). METHODS: Eligible patients were adults with recurrent supratentorial GBM; solitary tumor 1-5cm in diameter; ≥4 weeks after chemotherapy, bevacizumab or study drug; adequate organ function; KPS > 70%; and positive anti-polio titer. The original two-step continual reassessment method dose escalation was amended to treat 6 patients at level 2 after observing prolonged steroid use in 5 of 7 patients treated on levels 3 to 5. RESULTS: Thus far, 13 patients have been treated (1 each levels 1 and 3, 5 level 2, 2 level 4, 4 level 5). One dose limiting toxicity was observed, grade 4 intracranial hemorrhage at catheter removal (level 5). Adverse events possibly related to study include: hemiparesis (grade 3, n = 3; grade 2, n = 1; grade 1, n = 1); lymphopenia (grade 3, n = 1); seizure (grade 3, n = 1; grade 2, n = 1; grade 1, n = 2); dysphasia (grade 2, n = 1; grade 1, n = 1); lethargy (grade 1, n = 4); headache (grade 2, n = 1; grade 1, n = 2); one each of grade 2 diarrhea, paresthesia and hyperbilirubinemia; and one each of grade 1 fever, cough, nasal congestion, memory impairment, thrombocytopenia, anemia, nausea and vomiting. Ten patients remain alive, with pts #1 and #2 now 25 and 24 months post-PVSRIPO, respectively. Patients on levels 3 to 5 requiring prolonged steroid use were initiated on bevacizumab, allowing tapering down steroids. CONCLUSION: Infusion of PVSRIPO via CED is safe thus far and encouraging efficacy results are observed. Enrollment will be completed for the meeting.

Abstract CT416: Intratumoral administration of an Oncolytic Polio/Rhinovirus Recombinant (PVSRIPO) in recurrent glioblastoma (GBM): Preliminary results of the Phase I clinical trial

Abstract Background: PVSRIPO is the live attenuated, oral (SABIN) serotype 1 poliovirus vaccine containing a heterologous internal ribosomal entry site stemming from human rhinovirus type 2. An oncofetal cell adhesion molecule and tumor antigen widely expressed ectopically in malignancy, nectin-like molecule-5, is recognized by PVSRIPO. We report the preliminary results of a phase I clinical trial evaluating the intratumoral administration via convection-enhanced delivery (CED) of PVSRIPO. Methods: Eligibility criteria for adult patients included: recurrent supratentorial GBM; 1-5 cm in diameter; ≥1cm away from the ventricles; ≥4 weeks after chemotherapy, bevacizumab (BEV) or study drug; adequate organ function; KPS >70%; and positive anti-poliovirus titer. Dose was rapidly escalated using a two-step continual reassessment method with anticipated accrual of 1 patient each on dose levels 1-4, and up to 21 patients at dose level 5. Results: Thus far, ten patients have been treated (1 each at levels 1 and 3, 2 at level 2, 2 at level 4, 4 at level 5). One dose limiting toxicity (patient #8) was observed at level 5, a grade 4 intracranial hemorrhage at the time of catheter removal, which required de-escalation to level 4. Grade 3 adverse events possibly related to study include hemiparesis (n=1) and lymphopenia (n=1). No grade 5 study related adverse events were observed. Eight patients remain alive, with two patients now 20 and 19 months post PVSRIPO, respectively. Two patients having previously failed BEV died six months post-infusion after initiating hospice care due to persistence of baseline neurologic deficits. After observing prolonged steroid use in 5 of 7 patients treated on dose levels 3 to 5 and after results of new immunogenic analysis became available, it was agreed upon that dose level 2 is probably the optimal dose level. The study has been amended to treat a total of 6 patients at dose level 2. Conclusion: Infusion of PVSRIPO via CED in the clinical setting is safe thus far and observed efficacy outcomes are intriguing. Dose expansion at dose level 2 is ongoing. Citation Format: Annick Desjardins, J H. Sampson, K B. Peters, T Ranjan, G Vlahovic, S Threatt, J E. Herndon, S Boulton, D Lally-Goss, F McSherry, A Friedman, H S. Friedman, D D. Bigner, M Gromeier. Intratumoral administration of an Oncolytic Polio/Rhinovirus Recombinant (PVSRIPO) in recurrent glioblastoma (GBM): Preliminary results of the Phase I clinical trial. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT416. doi:10.1158/1538-7445.AM2014-CT416

Application of FCS in studies of rhinovirus receptor binding and uncoating.

Fluorescence correlation spectroscopy (FCS) allows determining diffusion and relaxation properties of fluorescent molecules. It requires only extremely small amounts of sample, down to picomolar concentrations, in an effective analysis volume of a few femtoliters. In essence, FCS determines the autocorrelation of fluorescence fluctuations caused by single labeled molecules passing through the confocal volume of a microscope equipped with a suitable detector; it permits investigating interactions of (macro)molecules, even in single cells. We present an FCS protocol for exploring, under in vitro conditions, the dynamic processes that take place during the early steps of virus infection. We cover two important issues of rhinovirus research, the kinetics of directional RNA release, and virus-receptor interactions exemplified by using human rhinovirus type A2 (HRV-A2) as a model.

Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells.

Mitogen-activated protein kinase-interacting kinase regulates mTOR/AKT signaling and controls the serine/arginine-rich protein kinase-responsive type 1 internal ribosome entry site-mediated translation and viral oncolysis.

Translation machinery is a major recipient of the principal mitogenic signaling networks involving Raf-ERK1/2 and phosphoinositol 3-kinase (PI3K)-mechanistic target of rapamycin (mTOR). Picornavirus internal ribosomal entry site (IRES)-mediated translation and cytopathogenic effects are susceptible to the status of such signaling cascades in host cells. We determined that tumor-specific cytotoxicity of the poliovirus/rhinovirus chimera PVSRIPO is facilitated by Raf-ERK1/2 signals to the mitogen-activated protein kinase (MAPK)-interacting kinase (MNK) and its effects on the partitioning/activity of the Ser/Arg (SR)-rich protein kinase (SRPK) (M. C. Brown, J. D. Bryant, E. Y. Dobrikova, M. Shveygert, S. S. Bradrick, V. Chandramohan, D. D. Bigner, and M, Gromeier, J. Virol. 22:13135-13148, 2014, doi:http://dx.doi.org/10.1128/JVI.01883-14). Here, we show that MNK regulates SRPK via mTOR and AKT. Our investigations revealed a MNK-controlled mechanism acting on mTORC2-AKT. The resulting suppression of AKT signaling attenuates SRPK activity to enhance picornavirus type 1 IRES translation and favor PVSRIPO tumor cell toxicity and killing. Oncolytic immunotherapy with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES, is demonstrating early promise in clinical trials with intratumoral infusion in recurrent glioblastoma (GBM). Our investigations demonstrate that the core mechanistic principle of PVSRIPO, tumor-selective translation and cytotoxicity, relies on constitutive ERK1/2-MNK signals that counteract the deleterious effects of runaway AKT-SRPK activity in malignancy.

ONCOLYTIC POLIOVIRUS IMMUNOTHERAPY OF GLIOBLASTOMA

BACKGROUND: With the exception of Burkitt lymphoma, almost all solid cancers are susceptible to infection with poliovirus, due to widespread ectopic expression of the poliovirus receptor, the onco-fetal cell adhesion molecule Necl5/CD155. We engineered a profoundly CNS-incompetent and genetically stable variant of polio through recombination of the cognate internal ribosomal entry site (IRES) with its counterpart from human rhinovirus type 2. The resulting PVSRIPO chimera is incapable of causing poliomyelitis or encephalitis in non-human primates or human subjects, even after high-dose intracerebral inoculation, but retains excellent cytotoxic properties in malignant cells. This is due to constitutively active signal transduction pathways via Raf-Erk MAPKs to the MAPK-interacting kinase, Mnk, and the translation apparatus. METHODS: Raf-Erk-Mnk signals favor translation of mRNAs with highly structured, encumbered 5' untranslated regions, typical for mRNAs encoding potent biological response modifiers, survival factors, growth factors or oncogenes. They afford an enormous advantage to PVSRIPO translation, causing irreversible lethal cytotoxicity as early as 60 min after infection. PVSRIPO infection of GBM produces drastic, violent cytotoxicity and a wide range of pro-inflammatory and immunogenic host responses. These are due to virally-induced ‘immunogenic cell death’, forceful activation of innate antiviral type 1 interferon responses, non-lethal infection and pro-inflammatory stimulation of tumor-associated macrophages/dendritic cells and stimulation of cytotoxic T-cell responses directed against the tumor. RESULTS: A currently accruing Phase-1 clinical trial of PVSRIPO with intratumoral, CED-mediated infusion in recurrent GBM is showing promise with the first patients experiencing durable complete clinical and near-complete radiographic responses >20 months post PVSRIPO infusion. The key to PVSRIPO oncolytic immunotherapy is the early acute phase of viral cytotoxicity after intratumoral infusion. Unhinged translation control, enabling unfettered viral, IRES-mediated initiation, is independent of the notorious intratumoral and inter-individual heterogeneity of advanced, treatment-refractory cancers. To define the molecular mechanism of PVSRIPO-mediated immunogenic cell death, we unraveled a signaling network centered on Raf-Erk-Mnk signals and their control over the Ser-Arg-rich protein kinase. CONCLUSIONS: Provided a solid mechanistic rationale exists for their use, oncolytic viruses such as PVSRIPO may induce tumor-cytotoxic, immune-modulatory and immune-stimulatory effects with an extent and biological range that are difficult to achieve with any other type of anti-cancer agent. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

ONCOLYTIC POLIO/RHINOVIRUS RECOMBINANT (PVSRIPO) IN RECURRENT GLIOBLASTOMA (GBM): FIRST PHASE I CLINICAL TRIAL EVALUATING THE INTRATUMORAL ADMINISTRATION

BACKGROUND: PVSRIPO is the live attenuated, oral (SABIN) serotype 1 poliovirus vaccine containing a heterologous internal ribosomal entry site stemming from human rhinovirus type 2. PVSRIPO recognizes nectin-like molecule-5, an oncofetal cell adhesion molecule and tumor antigen widely expressed ectopically in malignancy. Within, we report on the ongoing phase I study evaluating the intratumoral convection-enhanced delivery (CED) of PVSRIPO. METHODS: Eligibility criteria for adult patients with recurrent supratentorial GBM included: 1-5 cm in diameter; ≥1cm away from the ventricles; ≥4 weeks after chemotherapy, bevacizumab (BEV) or study drug; adequate organ function; KPS >70%; and positive anti-poliovirus titer. Dose was rapidly escalated using a two-step continual reassessment method with anticipated accrual of 1 patient each on dose levels 1-4, and up to 21 patients at dose level 5. RESULTS: Thus far, ten patients have been treated (1 each at levels 1 and 3, 2 at level 2, 2 at level 4, 4 at level 5). One dose limiting toxicity (patient #8) was observed at level 5, a grade 4 intracranial hemorrhage at the time of catheter removal, which required de-escalation to level 4. Grade 2 and higher adverse events possibly related to study include: hemiparesis (grade 3, n = 1; grade 2, n = 1); lymphopenia (grade 3, n = 1); seizure (grade 2, n = 1); and one each of grade 2 diarrhea, paresthesia, dysphasia and hyperbilirubinemia. To date, 8 out of 10 patients are alive, with patients #1 and #2 now 20 and 19 months post PVSRIPO, respectively. Two BEV failure patients died six months post-infusion after hospice care was initiated due to persistence of baseline neurologic limitations. After observing prolonged steroid use in 5 of 7 patients treated on dose levels 3 to 5 and after results of new immunogenic analysis became available, it was agreed upon that dose level 2 is probably the optimal dose level. To date, the original study design has been amended to treat a total of 6 patients at dose level 2. CONCLUSIONS: Infusion of PVSRIPO via CED is safe thus far and observed efficacy outcomes are intriguing and warrant further clinical investigation. SECONDARY CATEGORY: Immunobiology & Immunotherapy.

Detection of Influenza and Other Respiratory Viruses Carried Out in the Influenza Project - Monitoring Vaccine Effectiveness (I-MOVE).

The project Influenza Vaccine Effectiveness-Monitoring (I-MOVE) is part of the European research carried out by the ECDC (European Center for Disease Prevention and Control), aimed at monitoring the effectiveness of vaccination in Europe during the growing incidence of flu and influenza-like illnesses in the coming epidemic seasons. Laboratory studies using molecular RT-PCR biology methods for detection of genetic material of influenza virus and other respiratory viruses were performed by Voivodeship Sanitary-Epidemiological Stations in Poland. The validation of the results of swabs taken from the nose and throat were carried out in the Department of Influenza Research, National Influenza Center in Warsaw. The study involved 210 samples from patients across Poland. Positive results were recorded for 72.4 % of the samples; influenza virus type A was detected in 43 and type B in 38 cases, whereas in 71 cases other respiratory viruses were detected, which included Human parainfluenza virus type 1–4; Human respiratory syncytial virus type A and B; Human coronavirus 229E/NL63, OC43; Human rhinovirus type A, B, and C; Human enterovirus; and Human adenovirus. The results show that although influenza viruses predominated in the 2010/2011 season in Poland, other flu-like viruses also abounded.

Detection of respiratory tract pathogens with molecular biology methods.

This paper describes the use in routine diagnosis of virological kit, which was designed to identify the 15 most common respiratory viruses in clinical specimens of nasopharyngeal aspirates, swabs, and bronchoalveolar lavage. It is a one-step multiplex RT-PCR system for the detection of influenza virus type A and type B, human respiratory syncytial virus type A, B; human adenovirus, human metapneumovirus, human coronaviruses 229E/NL63 and OC43, human parainfluenza type 1, 2, 3, human rhinovirus type A, B, human enterovirus, and bocavirus 1, 2, 3, 4. The article presents research conducted on the basis of swabs collected from patients who came to the Ear, Nose, and Throat Emergency Care Unit at the Department of Otolaryngology, Military Medical Institute in Warsaw, in February 2013. Due to the nature of work in an laryngological emergency ward, the material was collected only from those patients who reported problems associated with rhinitis or any dysfunction of the upper respiratory tract. The study shows that patients who came to seek laryngological assistance were usually infected with viruses having affinity for the airway epithelium.

Multiple Classes of Antiviral Agents Exhibit In Vitro Activity against Human Rhinovirus Type C

Human rhinovirus type C (HRV-C) is a newly discovered enterovirus species frequently associated with exacerbation of asthma and other acute respiratory conditions. Until recently, HRV-C could not be propagated in vitro, hampering in-depth characterization of the virus replication cycle and preventing efficient testing of antiviral agents. Herein we describe several subgenomic RNA replicon systems and a cell culture infectious model for HRV-C that can be used for antiviral screening. The replicon constructs consist of genome sequences from HRVc15, HRVc11, HRVc24, and HRVc25 strains, with the P1 capsid region replaced by a Renilla luciferase coding sequence. Following transfection of the replicon RNA into HeLa cells, the constructs produced time-dependent increases in luciferase signal that can be inhibited in a dose-dependent manner by known inhibitors of HRV replication, including the 3C protease inhibitor rupintrivir, the nucleoside analog inhibitor MK-0608, and the phosphatidylinositol 4-kinase IIIβ (PI4K-IIIβ) kinase inhibitor PIK93. Furthermore, with the exception of pleconaril and pirodavir, the other tested classes of HRV inhibitors blocked the replication of full-length HRVc15 and HRVc11 in human airway epithelial cells (HAEs) that were differentiated in the air-liquid interface, exhibiting antiviral activities similar to those observed with HRV-16. In summary, this study is the first comprehensive profiling of multiple classes of antivirals against HRV-C, and the set of newly developed quantitative HRV-C antiviral assays represent indispensable tools for the identification and evaluation of novel panserotype HRV inhibitors.

Virus shedding after human rhinovirus infection in children, adults and patients with hypogammaglobulinaemia

The shedding of human rhinovirus (HRV) after an acute, naturally acquired infection has not been described in detail. We determined the duration of HRV shedding in immunocompetent children and adults, and in patients with primary hypogammaglobulinaemia. Subjects with symptoms of respiratory tract infection, and their household contacts, were screened for HRV by reverse transcription PCR. They were followed by serial, self-collected nasal swab specimens until negative for HRV or infected by another HRV type. We followed 62 HRV infections in 54 subjects. The mean (95% CI) duration of HRV shedding was 11.4 (8.2–14.7) days in children, 10.1 (7.4–12.9) days in adults, and 40.9 (26.4–55.4) days in patients with hypogammaglobulinaemia (p <0.001). The duration of respiratory tract symptoms correlated with the duration of virus shedding (p 0.002). A new infection by another HRV type soon after the first episode was common. We conclude that the shedding times of HRV are relatively short in otherwise healthy individuals. In contrast, prolonged shedding over 28 days is frequent in patients with hypogammaglobulinaemia despite immunoglobulin replacement therapy.

Dose-finding and safety study of an oncolytic polio/rhinovirus recombinant against recurrent glioblastoma.

2094 Background: Current therapies for glioblastoma are limited by ineffective delivery beyond the blood-brain barrier, limited diffusion of macromolecules, and lack of tumor specificity. Sustained direct intracerebral infusion at slow flow rates [convection-enhanced delivery (CED)] can overcome delivery barriers. PVSRIPO is the live attenuated, oral (SABIN) serotype 1 poliovirus vaccine containing a heterologous internal ribosomal entry site stemming from human rhinovirus type 2. PVSRIPO recognizes nectin-like molecule-5, an oncofetal cell adhesion molecule and tumor antigen widely expressed ectopically in malignancy. We report the results of an ongoing phase I study evaluating PVSRIPO when delivered by CED. Methods: Eligible on study are adult patients with: 1-5 cm of measurable supratentorial recurrent glioblastoma ≥1cm away from the ventricles; ≥4 weeks after chemotherapy, bevacizumab or study drug; adequate organ function; KPS ≥70%; and positive anti-poliovirus titer. PVSRIPO is delivered intratumorally by CED over 6.5 hours. PVSRIPO dose escalation is accomplished by increasing agent concentration, allowing flow-rate and infusion volume to remain constant. Two-step continual reassessment method is used for dose escalation, with one patient each treated on dose levels 1-4, and a possibility of up to 13 patients on dose level 5. Results: Thus far, a total of five patients have been treated on study. No related or unrelated grade 3 or higher adverse events have been observed. Grade 1 adverse events possibly related to the study drug or procedure include one each of fever, cough, nasal congestion, vomiting, headache, hemiparesis, and lethargy. Grade 2 adverse events include one each of diarrhea and seizure. Patient #1 had failed bevacizumab prior to enrollment and remains disease free more than 9 months post PVSRIPO. Two more patients are disease free 8+ and 2+ months post treatment, respectively. One patient had pathology confirmed disease recurrence two months post treatment and one patient came off study due to clinical decline four months post treatment. Conclusions: Infusion of PVSRIPO via CED is safe thus far and encouraging efficacy results are observed. Updated results will be presented. Clinical trial information: NCT 01491893.

Polypyrimidine tract binding protein-1 (PTB1) is a determinant of the tissue and host tropism of a human rhinovirus/poliovirus chimera PV1(RIPO).

The internal ribosomal entry site (IRES) of picornavirus genomes serves as the nucleation site of a highly structured ribonucleoprotein complex essential to the binding of the 40S ribosomal subunit and initiation of viral protein translation. The transition from naked RNA to a functional "IRESome" complex are poorly understood, involving the folding of secondary and tertiary RNA structure, facilitated by a tightly concerted binding of various host cell proteins that are commonly referred to as IRES trans-acting factors (ITAFs). Here we have investigated the influence of one ITAF, the polypyrimidine tract-binding protein 1 (PTB1), on the tropism of PV1(RIPO), a chimeric poliovirus in which translation of the poliovirus polyprotein is under the control of a human rhinovirus type 2 (HRV2) IRES element. We show that PV1(RIPO)'s growth defect in restrictive mouse cells is partly due to the inability of its IRES to interact with endogenous murine PTB. Over-expression of human PTB1 stimulated the HRV2 IRES-mediated translation, resulting in increased growth of PV1(RIPO) in murine cells and human neuronal SK-N-MC cells. Mutations within the PV1(RIPO) IRES, selected to grow in restrictive mouse cells, eliminated the human PTB1 supplementation requirement, by restoring the ability of the IRES to interact with endogenous murine PTB. In combination with our previous findings these results give a compelling insight into the thermodynamic behavior of IRES structures. We have uncovered three distinct thermodynamic aspects of IRES formation which may independently contribute to overcome the observed PV1(RIPO) IRES block by lowering the free energy δG of the IRESome formation, and stabilizing the correct and functional structure: 1) lowering the growth temperature, 2) modifying the complement of ITAFs in restricted cells, or 3) selection of adaptive mutations. All three mechanisms can conceivably modulate the thermodynamics of RNA folding, and thus facilitate and stabilize the functional IRES structure.

Neuroattenuation of Vesicular Stomatitis Virus through Picornaviral Internal Ribosome Entry Sites

Vesicular stomatitis virus (VSV) is potent and a highly promising agent for the treatment of cancer. However, translation of VSV oncolytic virotherapy into the clinic is being hindered by its inherent neurotoxicity. It has been demonstrated that selected picornaviral internal ribosome entry site (IRES) elements possess restricted activity in neuronal tissues. We therefore sought to determine whether the picornavirus IRES could be engineered into VSV to attenuate its neuropathogenicity. We have used IRES elements from human rhinovirus type 2 (HRV2) and foot-and-mouth disease virus (FMDV) to control the translation of the matrix gene (M), which plays a major role in VSV virulence. In vitro studies revealed slowed growth kinetics of IRES-controlled VSVs in most of the cell lines tested. However, in vivo studies explicitly demonstrated that IRES elements of HRV2 and FMDV severely attenuated the neurovirulence of VSV without perturbing its oncolytic potency.

Persistent human rhinovirus type C infection of the lower respiratory tract in a pediatric cord blood transplant recipient

Persistent human rhinovirus type C infection of the lower respiratory tract in a pediatric cord blood transplant recipient

Homology Modeling and Analysis of Structure Predictions of the Bovine Rhinitis B Virus RNA Dependent RNA Polymerase (RdRp)

Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3Dpol). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3Dpol using a combination of different computational tools. Model structures of BRBV 3Dpol were verified for their stereochemical quality and accuracy. The BRBV 3Dpol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP) and pyrophosphate (PPi) bound FMDV 3Dpol (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3Dpol as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3Dpols is also substantiated by phylogeny analysis and root-mean square deviation (RMSD) between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3Dpol compared to FMDV 3Dpol, indicate that despite a very high homology to FMDV 3Dpol, BRBV 3Dpol may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable to Aphthovirus RdRps.

Sequence Analysis of a Cloned Human Rhinovirus Designed For Use in Human Inoculation Studies

Inoculation with human rhinovirus type 16 (HRV16) has been used extensively to study the pathogenesis of colds. Previous HRV16 inoculum stocks were grown from the nasal secretions of experimentally infected volunteers. To provide a genetically stable source of HRV and minimize the risk of infectious contaminants, we developed a new reverse genetics inoculum virus (RG-HRV16) from a proven inoculum stock (HRV16-KC). We hypothesized that our production method would produce a progeny HRV that is highly similar to that of the original HRV16-KC.