Reovirus RNA Research Articles

Reovirus RNA recombination is sequence directed and generates internally deleted defective genome segments during passage.

For viruses with segmented genomes, genetic diversity is generated by genetic drift, reassortment, and recombination. Recombination produces RNA populations distinct from full-length gene segments and can influence viral population dynamics, persistence, and host immune responses. Viruses in the Reoviridae family, including rotavirus and mammalian orthoreovirus (reovirus), have been reported to package segments containing rearrangements or internal deletions. Rotaviruses with RNA segments containing rearrangements have been isolated from immunocompromised and immunocompetent children and in vitro following serial passage at relatively high multiplicity. Reoviruses that package small, defective RNA segments have established chronic infections in cells and in mice. However, the mechanism and extent of Reoviridae RNA recombination are undefined. Towards filling this gap in knowledge, we determined the titers and RNA segment profiles for reovirus and rotavirus following serial passage in cultured cells. The viruses exhibited occasional titer reductions characteristic of interference. Reovirus strains frequently accumulated segments that retained 5' and 3' terminal sequences and featured large internal deletions, while similarly fragmented segments were rarely detected in rotavirus populations. Using next-generation RNA-sequencing to analyze RNA molecules packaged in purified reovirus particles, we identified distinct recombination sites within individual viral genome segments. Recombination junctions were frequently but not always characterized by short direct sequence repeats upstream and downstream that spanned junction sites. Taken together, these findings suggest that reovirus accumulates defective gene segments featuring internal deletions during passage and undergoes sequence-directed recombination at distinct sites.IMPORTANCE Viruses in the Reoviridae family include important pathogens of humans and other animals and have segmented RNA genomes. Recombination in RNA virus populations can facilitate novel host exploration and increased disease severity. The extent, patterns, and mechanisms of Reoviridae recombination and the functions and effects of recombined RNA products are poorly understood. Here, we provide evidence that mammalian orthoreovirus regularly synthesizes RNA recombination products that retain terminal sequences but contain internal deletions, while rotavirus rarely synthesizes such products. Recombination occurs more frequently at specific sites in the mammalian orthoreovirus genome, and short regions of identical sequence are often detected at junction sites. These findings suggest that mammalian orthoreovirus recombination events are directed in part by RNA sequences. An improved understanding of recombined viral RNA synthesis may enhance our capacity to engineer improved vaccines and virotherapies in the future.

The Effects of Human Reoviruses on Cancer Cells Derived from Hepatocellular Carcinoma Biopsies

Hepatocellular carcinoma (HCC) is the most common type of liver cancer around the world. Since this cancer is highly resistant to the existing treatments, we used a novel method, which selectively targets HCC cancer cells to improve the treatment process. As normal cells are resistant to reovirus replication, we used oncolytic reoviruses, which can infect, replicate in, and destroy cancer cells. In this study, the effects of oncolytic human reoviruses on cancer cells, derived from HCC biopsies, were investigated. First, reoviruses were purified. Then a plaque assay was performed to estimate the number of viruses and determine the multiplicity of infection (MOI). To evaluate the effects of reoviruses on cancer cells derived from HCC biopsies, replication of reovirus RNA, viral protein production, cytopathic effects (CPE), and cancer cell viability were assessed at different intervals post-infection. Replication of reovirus RNA and viral protein production were detected in cancer cells. Also, different levels of viral protein production, CPE, cytotoxicity, and cancer cell viability were observed at different intervals post-infection with human reoviruses. In contrast, normal human fibroblasts, which were used as negative control, remained unchanged. For the first time, the effects of human reoviruses on HCC biopsies were investigated. The results showed that human reoviruses could replicate in and destroy cancer cells derived from HCC biopsies. Overall, human reoviruses can be potentially used for the treatment of HCC.

A TaqMan-MGB real-time RT-PCR assay with an internal amplification control for rapid detection of Muscovy duck reovirus

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of Muscovy duck reovirus (MDRV) RNA in clinical samples is described. The assay is based on TaqMan-MGB technology, consisting of two primers and one probe labeled with the reporter dye 6-carboxyfluorescein that binds selectively to the sigma B-protein gene of MDRV. This technique also includes an Internal Positive Control (IPC). The real-time RT-PCR assay was able to detect MDRVs, whereas other common waterfowl-origin viral pathogens were not recognised by the established oligonucleotide set, thus showing that the test was specific for MDRV. The sensitivity of the assay was 2.83 × 101 copies/μL and was 100 times higher than that of the conventional RT-PCR. The variation coefficients of intra-assay and inter-assay were less than 1.5% which verified sufficient repeatability of this assay.The use of β-actin mRNA as an IPC in order not to reduce the efficiency of the assay was adopted. The detection for 100 clinical samples showed that the positive rate of the established TaqMan-MGB real-time RT-PCR method was 87% (87/100), while the positive rate of the conventional RT-PCR was 83% (83/100), with the coincidence rate was 97.14%. Sensitivity and positive rate for clinical samples of TaqMan fluorescent quantitative RT-PCR were higher than conventional RT-PCR. The high specificity, sensitivity, and rapidity TaqMan-MGB real-time RT-PCR assay with the use of IPC to monitor for false negative results can make this method suitable for the pathogenic surveillance and epidemiological investigation of MDRV infection.

Abstract 2272: Exploratory analysis of T cell repertoire dynamics upon systemic treatment with the oncolytic virus pelareorep in combination with pembrolizumab and chemotherapy in patients with advanced pancreatic adenocarcinoma

Abstract Background: Pelareorep is an immuno-oncolytic virus that induces an inflamed tumor phenotype in metastatic adenocarcinoma of the pancreas (MAP). Systemically delivered pelareorep in combination with chemotherapy achieves 1 & 2 year-survival rates of 46% & 24% in MAP pts, respectively. Analysis of tumor tissue from patients (pts) treated with pelareorep, chemotherapy and anti-PD-L1 have shown reovirus RNA and protein replication, T-cell infiltration, and upregulation of PD-L1, highlighting that effective T-cell recognition of tumor antigens may be critical to success for this combination therapy. Thus, we hypothesized that pelareorep in combination with chemo and pembrolizumab in pts with MAP would alter the peripheral T-cell repertoire, creating new T-cell clones via the release of novel neoantigens in addition to expanding existing T-cell clones. Methods: A phase 1b study enrolled 11 MAP pts who progressed after first-line treatment. Pts received pelareorep (4.5e10 TCID 50 IV, D1 & D2), plus pembrolizumab (2mg/kg IV, D8) plus either 1) 5-FU (LV (200 mg/m2 /5-FU 200 mg /m2 IV bolus, 5-FU 1200mg/m2 continuous IV infusion D1) or 2) gemcitabine (1000 mg/m2 IV, D1), or 3) irinotecan (125 mg/m2 IV, D1) q3w, until disease progression/unacceptable toxicity. DNA from peripheral blood mononuclear cells from nine patients at cycle 1 day 1 (C1D1) & C2D1 (approx. 3 weeks later) was analyzed using the immunoSEQ® Assay (Adaptive Biotechnologies, Seattle) sequencing the T-cell receptor beta chain region to interrogate changes in the T-cell repertoire. Results: The median Morisita index between C2D1 and C1D1 was 0.83 with 3 samples below 0.6, indicative of significant peripheral repertoire turnover. The median number of expanded clones equated to 45.7 per 100,000 cumulative templates; normal variation over 4 weeks is ~ 5-10 expanded clones. Strikingly, most (median: 86%) peripheral clonal expansion occurred in clones below the limit of detection at C1D1. Cox regression analysis revealed that high peripheral clonality correlates with progression free survival at C1D1 (p=0.01, HR=0.053). Moreover, high clonality correlates with overall survival at both C1D1 (p=0.013, HR=0.124) and C2D1 (p=0.010, HR=0.079). Conclusions: High levels of peripheral T-cell repertoire turnover occur between C1D1 and C2D1. Repertoire turnover is accompanied by significant clonal expansion, mostly by expansion of new clones (i.e. undetected in C1D1). Higher peripheral clonality is associated with better progression free survival at C1D1, and overall survival at C1D1 and C2D1. This research highlights the potential utility of T-cell clonality as a predictive and prognostic biomarker to pelareorep therapy and warrants further clinical investigation. Citation Format: Grey A. Wilkinson, Devalingam Mahalingam, Sukeshi Patel Arora, Paul A. Fields, Patrick Raber, Karol Cheetham, Matt Coffey. Exploratory analysis of T cell repertoire dynamics upon systemic treatment with the oncolytic virus pelareorep in combination with pembrolizumab and chemotherapy in patients with advanced pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2272.

THE PROBLEM OF CELL CULTURES CONTAMINATION WITH MAMMALIAN ORTHOREOVIRUSES

A mandatory requirement for cell cultures used in scientific researches and biomanufacturing is the absence of their contamination by viruses. We have described the case of adventitious isolation of mammalian orthoreovirus in the rhesus macaque embryo kidney cells MA-104. PCR analysis for the presence of reovirus RNA of all probable sources of reovirus (trypsin, fetal bovine serum, clinical samples, cell culture) revealed no viral RNA in any of the samples. An important condition for the activation of the reovirus reproduction in the MA-104 cells was the presence of trypsin in the culture medium. The obtained results underscore the urgency of control for the reovirus contamination of chemicals of animal origin and cell cultures. Since reoviruses are associated with human diseases, such control in pharmaceutical production is mandatory.

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions.

Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5' nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation.IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure.

P07.05 Intravenous delivery of oncolytic reovirus to brain tumours in patients to immunologically prime for sequential checkpoint blockade

Abstract Introduction: Therapies targeting T-cell inhibitory checkpoint signalling pathways are redefining cancer survival in many solid malignancies. Unfortunately only a minority of patients respond, with mounting evidence that tumour response and patient survival are significantly associated with the presence of tumour PD-L1 expression, which prevents immune activation in situ within the tumour. We hypothesised that pre-conditioning of the tumour immune microenvironment using targeted, virus-mediated interferon stimulation, would upregulate PD-L1 protein expression and improve the efficacy of sequential checkpoint blockade. Oncolytic viruses (OV) stimulate anti-tumour immunity, and represent a promising novel form of cancer immunotherapy. However almost all brain tumour studies to date administer OV via intra-lesion injection, following a largely untested belief that intra-venous (IV) administration will not deliver virus to this site, due to exclusion by the blood brain barrier. Materials and Methods: In this window-of-opportunity clinical study 9 patients were injected IV once with 1x1010 TCID50 of oncolytic human Orthoreovirus (reovirus) ahead of planned surgical resection of their brain tumour. Following resection, tumour tissue was analysed by RNAseq and virus presence assayed by IHC, in-situ-hybridisation, electron microscopy and qPCR. Virus regulated expression of PD1/PDL1 axis was determined in ex vivo brain tumour culture, and intracranial tumour models were used to assess combination treatment of OV and checkpoint therapy. Results: Here we show that IV injection of reovirus leads to infection of tumour cells both in high grade glioma and brain metastases. Reovirus σ3 protein and RNA were detected in all tumour samples by at least one detection method. RNAseq analysis indicated reovirus upregulates interferon-regulated gene expression in tumours, and we further show in stem-like cell lines and ex vivo primary human tumour cultures that the PD-1/PD-L1 axis is altered across both target tumour and immune effector cells via an IFN-mediated mechanism. Finally, in a pre-clinical glioma model we demonstrate significantly enhanced clinical therapy using combination treatment of PD-1 blockade and reovirus. CONCLUSION: These results support the development of novel combination, systemic immuno-virotherapy strategies for the treatment of both primary and secondary tumours in the brain.

Abstract A49: Systemic oncolytic reovirus for the treatment of primary and secondary brain tumors

Abstract Reovirus is a systemically delivered oncolytic agent with evidence of activity in both pre-clinical models and in early phase clinical trials. Reovirus has direct oncolytic activity against many human/murine tumor cells, as well as activating anti-tumor innate and adaptive immunity. Having previously shown that intravenously delivered reovirus selectively accesses colorectal cancer metastatic to the liver in patients, in this study we explored the potential of systemic reovirus for the treatment of both primary and secondary brain tumors. We first showed that intravenous reovirus can be detected in tumors implanted into the brains of immunocompetent mice. We then tested our current most potent reovirus-based therapy (systemic reovirus plus granulocyte-macrophage colony stimulating factor [GMCSF] in reovirus pre-immune mice), and showed effective therapy in immunocompetent murine models of primary glioma, and of both directly reovirus sensitive, and insensitive, models of melanoma brain metastases. We also found that addition of reovirus/GMCSF to clinical ‘standard of care’ (radiotherapy and temozolamide chemotherapy) significantly enhances survival. In parallel to these pre-clinical experiments, we have initiated an open-label, non-randomized study of intravenous reovirus administered to patients prior to planned surgery for recurrent high grade glioma or metastatic brain tumors, to test whether the data showing access of the agent to tumors in the brains in mice, also applies to humans. In total, 10 patients will be treated with a single infusion of 1x1010 TCID50 of reovirus, of which 9 have completed the study to date. The primary objective is the presence of reovirus in the resected tumors as assessed by immunohistochemistry, RNA in-situ hybridization and retrieval of infectious virions. Early analysis of the first 3 patients, comprising one glioblastoma multiforme, one grade 3 oligodendroglioma and one colorectal brain metastasis, has shown that all 3 resected tumors contained reovirus RNA and protein. There was also evidence for productive reovirus infection in 2 of the tumors. Within all patients to date, the only grade 3-4 adverse reaction has been neutropaenia in 1 patient. Further tissue analysis is ongoing, as is testing of blood samples from these patients to further charactize how reovirus is carried in the blood and protected from neutralizing antibodies. This clinical study shows, for the first time that an oncolytic virus, reovirus, infects and replicates in brain tumors following intravenous administration. Together with pre-clinical data showing the efficacy of systemic reovirus in combination with GMCSF/radiation/temozolamide, these findings support the future development of trials and combination studies using reovirus in patients with high grade gliomas and brain metastases. Citation Format: Adel Jebar, Liz Ilett, Tim Kottke, Emma West, Karen Scott, Simon Thomson, Matt Coffey, Gerard Nuovo, Susan Short, Richard Vile, Alan Melcher. Systemic oncolytic reovirus for the treatment of primary and secondary brain tumors. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A49.

In vitro anti-reovirus activity of kuraridin isolated from Sophora flavescens against viral replication and hemagglutination

In this study, we evaluated the anti-reovirus activity of kuraridin isolated from the roots of Sophora flavescens. In particular, we focused on whether this property is attributable to direct inhibition of reovirus attachment and/or inhibition of viral replication with the aid of time-of-addition (pre-treatment, simultaneous treatment, and post-treatment) experiments. No significant antiviral activity of kuraridin was detected in the pre-treatment assay. In the simultaneous assay, the 50% effective inhibitory concentrations (EC50) of kuraridin were 15.3–176.9 μM against human type 1–3 reoviruses (HRV1–3) and Korean porcine reovirus (PRV). Kuraridin completely blocked binding of viral sigma 1 protein to sialic acids at concentrations lower than 82.5 μM in the hemagglutination inhibition assay. Moreover, kuraridin inhibited HRV1–3 and PRV viral replication with EC50 values of 14.0–62.0 μM. Quantitative real-time PCR analysis disclosed strong suppression of reovirus RNA synthesis at the late stage (18 h) of virus replication by kuraridin. The viral yields of kuraridin-treated cells were significantly reduced at 24 h post-infection, compared with DMSO-treated cells. Our results collectively suggest that kuraridin inhibits virus adsorption and replication by inhibiting hemagglutination, viral RNA and protein synthesis and virus shedding, supporting its utility as a viable candidate antiviral drug against reoviruses.

Reovirus double-stranded RNA genomes and polyI:C induce down-regulation of hypoxia-inducible factor 1α

Reovirus has genomes consisting of 10-segmented double-stranded RNAs, and have received much attention as an oncolytic virus. A previous study reported that reovirus down-regulates hypoxia-inducible factor 1α (HIF-1α) protein levels following infection in tumor cells, which contributes to the antitumor effects of reovirus; however, the mechanism remains to be elucidated. In this study, we examined which virus component was involved in reovirus-mediated down-regulation of HIF-1α. Reovirus induced significant down-regulation of HIF-1α protein levels in not only reovirus-permissive tumor cells but also reovirus–resistant tumor cells. UV-inactivated reovirus also induced a reduction in HIF-1α protein levels. These data indicate that reovirus induces HIF-1α down-regulation independently of virus replication. Furthermore, transfection with not only reovirus genomes but also polyI:C efficiently induced HIF-1α down-regulation in a manner similar to reovirus, indicating that double-stranded reovirus RNA genomes are a key component for HIF-1α down-regulation. Reovirus-mediated HIF-1α down-regulation was inhibited when tumor cells were pretreated with inhibitors of cathepsins B and L, which play a crucial role in endo-lysosomal escape of virions to the cytoplasm. These data suggest that endo-lysosomal escape of reovirus genome into the cytoplasm is crucial for HIF-1α down-regulation; however, the retinoic acid-inducible gene-I (RIG-I) or interferon-β promoter stimulator-1 (IPS-1), which are involved in reovirus genome-induced innate immunity in the cytoplasm, did not play a crucial role in reovirus-mediated HIF-1α reduction.

Occurrence of reovirus infection in Muscovy ducks (Cairina moschata) in south western Poland.

During the summer 2012 an incidence of high mortality, above 44 percent, in two flocks of Muscovy ducklings in Poland was noted. The clinical signs included considerable weight loss and inability to walk. During the post-mortem evaluations dehydration and enteritis, gouty kidneys as well as hemorrhagic liver and spleen lesions were found. The laboratory diagnosis included agar gel precipitation assay (AGP) as well as polymerase chain reaction (PCR) or reverse transcription PCR for the presence of goose parvovirus (GPV), duck circovirus (DuCV), duck reovirus (DRV) and avian reovirus (ARV). Interestingly, the examinations performed by AGP showed partial reactivity of liver homogenates from Muscovy ducklings with chicken S1133 antiserum. The presence of duck reovirus RNA was also detected by real-time RT-PCR targeting the chicken reovirus sigma NS fragment, while the sequencing showed major similarity to chicken S1133, 1733, GX/2010/1 and TARV-MN2 reovirus strains. The virus sequence was also related to a previously isolated TH11 strain from Muscovy ducks in China. Further study is needed in order to explain the particular epidemiology of the reovirus infection of Muscovy ducklings.

Understanding The Differential Response Of Multiple Myeloma To Reovirus Treatment

IntroductionMultiple myeloma (MM) is a currently incurable malignancy arising from post germinal, terminally differentiated plasma cells. Despite the success of proteasome inhibitors and immune-modulating drugs such as bortezomib and thalidomide in the treatment of relapsed disease patients eventually become refractory to these drugs making new therapeutic agents a critical need.One potential new agent is Reovirus Serotype 3- Dearing Strain. While infection of immunocompetent hosts results in only minor respiratory illness it has been demonstrated that reovirus selectively replicates in and is cytopathic to Ras activated cells. This specificity is due at least in part to the inhibition of auto-phosphorylation of PKR. The minimal side effects and dependence of reovirus on Ras activation make it an interesting candidate for relapsed/refractory MM as it has been reported that at least 50% of relapsed MM patients have activating Ras mutations and most patients show activation at other Ras checkpoints including p38. While there has been limited investigation of reovirus treatment of MM cell lines there has been less analysis of resistance to reovirus treatment in MM. MethodsMM cell lines were screened via MTS assay to identify cell lines sensitive, intermediate, and resistant to reovirus treatment. Cell lines were then treated at a MOI of 10 or 25 for 9 and 24 hours and analyzed by immunohistochemistry (IHC) for p38, reoviral protein, and caspase-3 as well as in situ hybridization (ISH) and qRT-PCR for reoviral RNA. Total RNA isolates of sensitive, intermediate, and resistant cell lines were used to perform a screening analysis of mRNA, microRNA (miRNA), and long non-coding RNA to identify differential expression. In addition, expression of the reovirus high affinity receptor JAM-1 was assessed in these cell lines by western blot and IHC. Following this analysis mRNA, miRNA, and long non-coding RNA expression was analyzed in bone marrow samples from relapsed/refractory MM patients enrolled in a reovirus monotherapy clinical trial before and after treatment. These results were correlated with patient response and compared to the results from the cell lines. ResultsMTS results clearly identified several cell lines as being sensitive (RPMI), intermediate (H929), and resistant (OPM2) to reovirus treatment according to percent viability. IHC revealed over 98% positivity for p38 in all cell lines indicating Ras activation and permissibility to reovirus infection regardless of sensitivity. IHC also showed a higher percentage of cells positive for reoviral protein in sensitive and intermediate cell lines compared to resistant cell lines. The increased reoviral protein expression in these cell lines correlated with these lines having a higher percentage of caspase-3 positive cells compared to both the controls and the resistant cell lines. ISH demonstrated that the relative signal intensity of reoviral RNA was proportional to the sensitivity of each cell line, and these results were consistent with qRT-PCR results. ConclusionsDespite susceptibility to reovirus infection, as determined by p38 positivity, substantial variation was seen in response to reovirus treatment amongst cell lines. Given the correlation of cell line sensitivity with reoviral RNA levels and reoviral protein production it will be important to identify factors that drive these differences. Ongoing analysis is focused on determining the role of viral entry including expression of the reoviral receptor JAM-1 and subsequent viral trafficking and degradation. In addition, evaluation of differential expression of RNA molecules in cell lines and patient samples is ongoing as miRNA have been identified in the establishment of productive reoviral infections and cancer cell death (Nuovo et al. 2012). Disclosures:No relevant conflicts of interest to declare.Table 1All results are for cells treated at MOI 25 for 24 hours except % viability in which cells were treated at MOI 25 for 48 hours. Values for p38, reovirus protein and caspase-3 represent the percentage of positive cells by IHC. ISH reovirus RNA represents relative signal intensity with 100% being maximum intensity.Cell Line% Viabilityp38Reovirus ProteinCaspase-3 VirusCaspase-3 ControlISH Reovirus RNAAvg. Ct qRT-PCRRPMI2.599.245.359.037.39515.7H92946.398.69.338.517.69019.2OPM287.098.43.642.049.06028.4

Bio-distribution study of Reolysin® (pelareorep) through a single intravenous infusion in Sprague-Dawley rats

Numerous pre-clinical and clinical studies on reovirus have generated valuable information which supports the use of this orphan virus as an investigational drug for cancer treatment. Reolysin® (pelareorep) is a clinical formulation of the human Reovirus Type 3 Dearing strain. The clinical safety and efficacy of Reolysin® in humans is being tested on an assortment of cancer indications as a mono and/or combination therapy. Reovirus has many inherent characteristics that make it a potential candidate for virotherapy, including: the rapid and natural spread through the haematogenous route, the ability to overcome immunological barriers thereby reaching tumor sites, and being replication-competent. The purpose of this study was to elucidate the bio-distribution pattern of Reolysin® in healthy Sprague-Dawley rats. Following a single 15-min intravenous infusion via the tail vein in Sprague-Dawley rats, the levels of virus genome were determined in 16 organs/tissues by RT-qPCR (Reverse Transcriptase- Quantitative Polymerase Chain Reaction) over a 336 h (Day 15) incubation regime. Consistent with previous studies, maximal reovirus RNA levels were observed in the spleen; indicating its involvement in viral uptake and clearance, followed by heart, ovaries, tail (infusion site), liver and lungs. All the organs/tissues demonstrated unquantifiable levels of reovirus genome at the end of incubation, suggesting substantial to complete viral clearance. Several studies in the last decade have described the use of reovirus for treating ovarian cancers. An increase of reovirus genome in ovaries at 24 h post infection was noted. The results will aid in the design of additional exploratory clinical trials for Reolysin®.

Inhibition of the replication of grass carp reovirus in CIK cells with plasmid-transcribed shRNAs

Two DNA constructs targeting the grass carp reovirus (GCRV) RNA dependent RNA polymerase (RdRp) gene and outer capsid protein (OCP) gene that were each 64 bp in length were synthesized chemically and cloned into pSilencer2.1-U6 neo plasmid, named pSi-RdRp1286 and pSi-OCP117, respectively. After transfection of pSi-RdRp1286 and pSi-OCP117 plasmids into CIK cells, the inhibition of GCRV replication in the cells were detected by observing cytopathic effect (CPE), quantitating virus titers (TCID 50/mL) and real-time quantitative RT-PCR analysis of viral RdRp and OCP genes. Five days after the cells were challenged with GCRV, both pSi-RdRp1286 and pSi-OCP117 reduced the viral titers by 5.47 lgTCID 50/mL and 4.37 lgTCID 50/mL, respectively. Compared to the positive control, CPE induced by GCRV in transfected cells was delayed and significantly less. Furthermore, the real-time quantitative RT-PCR analysis of the viral RdRp gene and OCP gene showed that the targeted gene expression were reduced by 89% and 73%, respectively. These results proved that the plasmid-transcribed shRNAs could inhibit effectively GCRV replication in CIK cells. These shRNAs provide potential tools for inhibiting GCRV infection and replication both in vitro and in vivo.

Modification of mammalian reoviruses for use as oncolytic agents

The Reoviridae are a family of viruses with a non-enveloped icosahedral capsid and a segmented double-stranded RNA genome. Prototypes of the mammalian Orthoreoviruses have been isolated from human respiratory and enteric tracts and are not associated with human disease. One of these, human reovirus type 3 Dearing (T3D), usually serves as a model for the family. In the last decade the mammalian Orthoreoviruses, especially T3D, have been evaluated as oncolytic agents in experimental cancer therapy. This is based on the observation that reoviruses induce cell death and apoptosis in tumor cells, but not in healthy non-transformed cells. Several clinical trials have been initiated in Canada, the USA, and the UK, to study the feasibility and safety of this approach. Due to the segmented structure of their double-stranded RNA genomes genetic modification of Reoviridae has been notoriously difficult. Several techniques have been described recently that facilitate the genetic modification of reovirus genomes. The basis for reverse genetics of reovirus is the discovery in 1990 that reovirus RNA is infectious. Subsequently, it took ten years before a foreign gene was introduced into the reovirus genome. Here we review the methods for reovirus modification and their use for generating new reovirus-derived oncolytic agents.

1235 A phase-I study of the combination of intravenous reovirus (REOLYSIN ®) and gemcitabine in patients with advanced cancer

A low-copy component of mammalian reovirus particles is μ2, an 83-kDa protein encoded by the M1 viral genome segment and packaged within the viral core. Previous studies have identified μ2 as a nucleoside triphosphate phosphohydrolase (NTPase) as well as an RNA 5′-triphosphate phosphohydrolase (RTPase), putatively involved in reovirus RNA synthesis and/or 5′-capping. Other studies have identified μ2 as a microtubule-binding protein, which also associates with the viral factory matrix protein μNS and thereby anchors the factories to cellular microtubules during infections by most reovirus strains. To extend studies of μ2 functions during infection, we tested a small interfering RNA (siRNA) directed against the M1 plus-strand RNAs of reovirus strains Type 1 Lang (T1L) and Type 3 Dearing (T3D). The siRNA strongly suppressed μ2 expression by either strain and reduced infectious yields in a strain-dependent manner. This first strain difference was genetically mapped to the M1 genome segment and tentatively assigned to a single μ2 sequence polymorphism, Pro/Ser208, which also determines a T1L–T3D strain difference in microtubule association. The siRNA-based defect in μ2 expression was rescued by plasmids, containing silent mutations in the siRNA-targeted sequence, which encoded either T1L or T3D μ2, but the growth defect was rescued only by T1L μ2. This second strain difference was also mapped to Pro/Ser208, in that swapping this one residue between T1L and T3D μ2 reversed the rescue phenotypes. Thus, the T1L–T3D strain difference in μ2–microtubule association was correlated not only with the extent of reduction in infectious yields by the siRNA but also with the extent of rescue by plasmid-derived μ2. In addition, the rescue capacity of T1L μ2 was abrogated by nocodazole treatment, providing independent evidence for the importance of μ2–microtubule association in plasmid-based rescue. In two separate cases, the results revealed functional differences between virus- and plasmid-derived μ2. Ala substitutions within the NTP-binding motif of T1L μ2 also abrogated its rescue capacity, suggesting that the NTPase or RTPase activity of μ2 is additionally required for effective viral growth.

Proteolytic Disassembly Is a Critical Determinant for Reovirus Oncolysis

See page 1406Mammalian ortheoreoviruses are currently being investigated as novel cancer therapeutics, but the cellular mechanisms that regulate susceptibility to reovirus oncolysis remain poorly understood. In this study, we present evidence that virion disassembly is a key determinant of reovirus oncolysis. To penetrate cell membranes and initiate infection, the outermost capsid proteins of reovirus must be proteolyzed to generate a disassembled particle called an infectious subviral particle (ISVP). In fibroblasts, this process is mediated by the endo/lysosomal proteases cathepsins B and L. We have analyzed the early events of infection in reovirus-susceptible and -resistant cells. We find that, in contrast to susceptible glioma cells and Ras-transformed NIH3T3 cells, reovirus-resistant cancer cells and untransformed NIH3T3 cells restrict virion uncoating and subsequent gene expression. Disassembly-restrictive cells support reovirus infection, as in vitro-generated ISVPs establish productive infection, and pretreatment with poly(I:C) does not prevent infection in cancer cells. We find that the level of active cathepsin B and L is increased in tumors and that disassembly-restrictive glioma cells support reovirus oncolysis when grown as a tumor in vivo. Together, these results provide a model in which proteolytic disassembly of reovirus is a critical determinant of susceptibility to reovirus oncolysis.

Silencing and complementation of reovirus core protein μ2: Functional correlations with μ2–microtubule association and differences between virus- and plasmid-derived μ2

A low-copy component of mammalian reovirus particles is μ2, an 83-kDa protein encoded by the M1 viral genome segment and packaged within the viral core. Previous studies have identified μ2 as a nucleoside triphosphate phosphohydrolase (NTPase) as well as an RNA 5′-triphosphate phosphohydrolase (RTPase), putatively involved in reovirus RNA synthesis and/or 5′-capping. Other studies have identified μ2 as a microtubule-binding protein, which also associates with the viral factory matrix protein μNS and thereby anchors the factories to cellular microtubules during infections by most reovirus strains. To extend studies of μ2 functions during infection, we tested a small interfering RNA (siRNA) directed against the M1 plus-strand RNAs of reovirus strains Type 1 Lang (T1L) and Type 3 Dearing (T3D). The siRNA strongly suppressed μ2 expression by either strain and reduced infectious yields in a strain-dependent manner. This first strain difference was genetically mapped to the M1 genome segment and tentatively assigned to a single μ2 sequence polymorphism, Pro/Ser208, which also determines a T1L–T3D strain difference in microtubule association. The siRNA-based defect in μ2 expression was rescued by plasmids, containing silent mutations in the siRNA-targeted sequence, which encoded either T1L or T3D μ2, but the growth defect was rescued only by T1L μ2. This second strain difference was also mapped to Pro/Ser208, in that swapping this one residue between T1L and T3D μ2 reversed the rescue phenotypes. Thus, the T1L–T3D strain difference in μ2–microtubule association was correlated not only with the extent of reduction in infectious yields by the siRNA but also with the extent of rescue by plasmid-derived μ2. In addition, the rescue capacity of T1L μ2 was abrogated by nocodazole treatment, providing independent evidence for the importance of μ2–microtubule association in plasmid-based rescue. In two separate cases, the results revealed functional differences between virus- and plasmid-derived μ2. Ala substitutions within the NTP-binding motif of T1L μ2 also abrogated its rescue capacity, suggesting that the NTPase or RTPase activity of μ2 is additionally required for effective viral growth.

Follow up study on viruses associated with SARS among the SARS patients

To study the existence status of the SARS-CoV, retrovirus, and the poliovirus in the bodies of the patients with SARS and the possible relationship between the three viruses and SARS. The clinical specimens of the nasopharyngeal swabs, sputum (or saliva), urine, fecal specimens were collected on three consecutive days from 8 patients with SARS 2 years after the recovery from SARS. SARS-CoV, reovirus and poliovirus RNA was detected by using reverse transcription (RT)-PCR; IgG antibody to the poliovirus type 1 and 3 and the antibody to SARS-CoV were determined using enzyme linked immunosorbent assay (ELISA). All the specimens were negative for SARS-CoV and reovirus by RT-PCR, but the fecal specimens from 4 persons were positive for poliovirus. The sequences of these poliovirus were highly homologous to that of human poliovirus type 1 strain sabin 1 genome at nucleotide level, but back mutations have occurred in the primary attenuating mutation sites at nucleotide position 480 (G --> A) in the 5' UTR and the nucleotide position 2795 (A --> G). No SARS-CoV, reovirus, and poliovirus were found in the normal controls. Three serum specimens were positive for the antibody to SARS-CoV. The IgG antibody to poliovirus were detected in 4 SARS patients and 23 healthy persons. No positive results for antibody to SARS-CoV were detected in the 25 healthy persons. The positive rate of the poliovirus antibody in the serum of SARS patients 2 years after recovery was significantly different from that of the normal controls, and the positive rate of poliovirus in the fecal specimens was still very high, and more importantly back mutations have occurred in the attenuating mutation sites at nucleotide position which plays an important role in the poliomyelitis.

Detection and identification of avian, duck, and goose reoviruses by RT-PCR: goose and duck reoviruses are part of the same genogroup in the genus Orthoreovirus

A reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of avian, duck, and goose reovirus (ARV, DRV, and GRV) RNA from cell culture supernatant and clinical samples was established. Based on multiple sequence alignment, a pair of degenerate primers was selected and synthesized. The amplified, cloned, and sequenced 598-base-pair products from the sigmaA-encoding gene fragment from 16 isolates (ranging over 30 years) indicated that the primer regions were well conserved. The sensitivity of this method was determined to be 10(-2) PFU. The specificity of the RT-PCR method was determined by testing specimens containing avian influenza A viruses, Newcastle disease virus, and infectious bronchitis virus, all of which yielded negative results with no discernible background. The efficiency of the system for detection of ARV, DRV, and GRV directly in 71/83 clinical samples was confirmed. The nucleotide sequence analysis indicated that DRV and GRV isolated from China in different locales and years were closely related, showing 97.4-100% homology to each other, but with only 86.7-88.5% identity to DRV 89026. The nucleotide and amino acid sequence identities in the amplified sigmaA-encoding gene were 74.2-78.4% and 86.9-92.0%, respectively, between duck/goose and chicken species. Phylogenetic analysis indicated that GRV and DRV aggregated into the same specified genogroup within subgroup II of the genus Orthoreovirus and are more closely related to ARV than to Nelson Bay virus. Overall, this study developed a sensitive and specific technique for the identification ARV, DRV, and GRV, and sequencing analysis has enhanced our understanding of the evolutionary relationship between ARV, DRV, and GRV.