Pteropine Orthoreovirus(PRV7S)does not Establish Persistent Infection in NP460 Cells but with Inflammatory Response Triggered

Pteropine orthoreovirus(PRV)is a nonenveloped double-stranded RNA(dsRNA)virus of the genus Orthoreovirus under the family Reoviridae.The PRV genome is composed of 10 dsRNA segments,including three large(L)segments,three medium(M)segments,and four small(S)segments surrounded by an icosahedral capsid[1].

禽类呼肠孤病毒(Avian reoviruses)感染

1病原学 1.1病毒特征禽类呼肠孤病毒为无囊膜,呈球形,双层衣壳和二十面对称的dsRNA病毒.粒子直径约60～80 nm,外层衣壳上有92颗中空的颗粒.在CsCl梯度离心中,感染病毒子的密度为1.29～1.30g/ml,无感染性空衣壳为1.29～1.30g/ml,病毒芯髓为1.44 g/ml.纯化病毒只含有RNA和蛋白质,平均含量分别为18.7%和81.3%.病毒在胞浆内复制,有时呈晶格排列.对热、pH 3和DNA代谢抑制物有抵抗力.MgCl2能增强病毒对热的稳定性,但当浓度太大时反而促其灭活.70%乙醇和0.5%有机碘可灭活病毒.一般无血凝活性. ……

High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells

Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid,which was named as pR/GCRV-VP7,was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover,the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.

Expression of Outer Capsid Protein VP5 of Grass Carp Reovirus in E.coli and Analysis of its Immunogenicity

Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.

Molecular Characterization of Nonstructural Protein NS38 of Grass Carp Reovirus

Viral nonstructural proteins in both enveloped and non-enveloped viruses play important roles in viral replication. Protein NS38 of Grass carp reovirus (GCRV), has been deduced to be a non-structural protein, and, consistent with other reoviruses, is considered to cooperate with the NS80 protein in viral particle assembly. To investigate the molecular basis of the role of NS38, a complete protein was expressed in E.coli for the first time. It was found that there is a better expression of NS38 induced with IPTG at 28 ℃ rather than 37 ℃. In addition, the antiserum of NS38 prepared with purified fusion protein and injected into rabbit could be used for detecting NS38 protein expression in GCRV infected cell lysate, while there is not any reaction crossed with purified virus particle, confirming NS38 is not a component of the viral structural protein. The result reported in this study will provide evidence for further viral protein-protein and protein-RNA interaction in dsRNA viruses replication.

The VP2 protein of grass carp reovirus(GCRV) expressed in a baculovirus exhibits RNA polymerase activity

The double-shelled grass carp reovirus （GCRV） is capable of endogenous RNA transcription and processing.Genome sequence analysis has revealed that the protein VP2,encoded by gene segment 2 （S2）,is the putative RNA-dependent RNA polymerase （RdRp）.In previous work,we have ex-pressed the functional region of VP2 that is associated with RNA polymerase activity （denoted as rVP2390-900） in E.coil and have prepared a polyclonal antibody against VP2.To characterize the GCRV RNA polymerase,a recombinant full-length VP2 （rVP2） was first constructed and expressed in a baculovirus system,as a fusion protein with an attached His-tag.Immunofluorescence （IF） assays,together with immunoblot （IB） analyses from both expressed cell extracts and purified Histagged rVP2,showed that rVP2 was successfully expressed in Sf9 cells.Further characterization of the replicase activity showed that purified rVP2 and GCRV particles exhibited poly（C）-dependent poly（G） polymerase activity.The RNA enzymatic activity required the divalent cation Mg2＋,and was optimal at 28 ℃.The results provide a foundation for further studies on the RNA polymerases of aquareoviruses during viral transcription and replication.

Clinical development of reovirus for cancer therapy:An oncolytic virus with immune-mediated antitumor activity

Reovirus is a double-stranded RNA virus with demonstrated oncolysis or preferential replication in cancer cells. The oncolytic properties of reovirus appear to be dependent, in part, on activated Ras signaling. In addition, Ras-transformation promotes reovirus oncolysis by affecting several steps of the viral life cycle. Reovirusmediated immune responses can present barriers to tumor targeting, serve protective functions against reovirus systemic toxicity, and contribute to therapeutic efficacy through antitumor immune-mediated effects via innate and adaptive responses. Preclinical studies have demonstrated the broad anticancer activity of wild-type, unmodified type 3 Dearing strain reovirus（Reolysin） across a spectrum of malignancies. The development of reovirus as an anticancer agent and available clinical data reported from 22 clinical trials will be reviewed.

High-resolution 3D Structures Reveal the Biological Functions of Reoviruses

Viruses in the family Reoviridae are non-enveloped particles comprising a segmented double-stranded RNA genome surrounded by a two-layered or multi-layered icosahedral protein capsid.These viruses are classified into two sub-families based on their particle structural organization.Recent studies have focused on high-resolution three-dimensional structures of reovirus particles by using cryo-electron microscopy (cryo-EM) to approach the resolutions seen in X-ray crystallographic structures.The results of cryo-EM image reconstructions allow tracing of most of the protein side chains,and thus permit integration of structural and functional information into a coherent mechanism for reovirus assembly and entry.

Suppression of RNA Interference Pathway in vitro by Grass Carp Reovirus

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined. The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK). The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay. GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells, no GCRV-specific siRNA could be detected. Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene. These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway, unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.

Establishment of Universal RT-PCR Detection Method for Duck Reovirus

This study was conducted to rapidly detect clinical infection condition of duck reovirus. A pair of specific primers was designed according to gene se- quence of σC protein of duck reovirus, and a specific RT-PCR detection method of duck reovirus was established with genome of duck reovirus as template. Differ- ent samples were collected from ducks infected by suspected reovirus in Jiangsu Province and subjected to PCR detection. The results showed that the established RT-PCR method could specifically amplify the 438 bp sequence of the conservative region of σC gene, and detec the DNA of duck rcovirus as low as 1gf, with a detection rate of 100%. The RT-PCR method could be used for rapid clinical diagnosis of duck reovirus.

Effect of Maternal Antibodies on the Pathogenesis of Avian Reovirus Infections in Broiler Chickens Using Real-Time Reverse Transcriptase Polymerase Chain Reaction

The effect of maternal antibodies on the pathogenesis of avian reovirus （ARV） was studied in commercial and specific pathogen free broilers （SPF） using a real-time reverse transcriptase （RT）-polymerase chain reaction （PCR） assay, along with the incidence and severity of morbidity, mortality, and gross lesions. ARV RNA was detected in cloacal swabs in both bird groups from the first day throughout the 21 days experiment. Commercial broiler chickens, which had high maternal antibodies against ARV, showed minimum clinical signs, gross lesions, and lower numbers of birds with viral RNA excretion, whereas specific pathogen free （SPF） broiler chickens, which did not have antibody against ARVs, had 30% mortality, more severe signs, and higher numbers of birds excreting viral RNA. The highest peak of SPF birds excreting viral RNA occurred during the time of maximum mortality. The protective effect of maternal antibody on ARV pathogenesis in broiler chickens correlated with the detection of ARV RNA using the real-time RT-PCR.

Development of Oncolytic Reovirus for Cancer Therapy

Reovirus, a double-stranded RNA virus, can infect many types of cancer cells and cause oncolysis. Mammalian reovirus has exhibited promising anticancer activity in clinical trials and holds great advantages and promise as an anticancer agent. Reovirus is not associated with any serious human diseases, naturally targets and destroys tumors, and lacks the DNA synthesis stage, thus avoiding potential DNA insertion mutations. This review discusses the properties of reovirus related to oncolysis and the mechanisms of oncolytic selection, and summarizes the preclinical and clinical studies that have led to the current Phase III trial. In addition, three major challenges in the development of reovirus-mediated oncolytic therapy are discussed. These are: the mechanisms of reovirus oncolysis remain to be fully characterized;the host immune responses should be manipulated to enhance viral anti-tumor effects;and the efficacy of reovirus oncolysis may be further improved by developing new vectors and studying other double-stranded RNA viruses.

Construction of Short Hairpin RNA Vector with σNS & σC Genes of Avian Reovirus and Determination of Interference Effect

[ Objective] The aim was to explore novel method for treatment of Avian Reovirus. [ Method] According to the design principle of siRNA target sequences, siRNA templates were designed and synthesized and then cloned into the shRNA expression vector, namely, pSilencer-CMV 4.1 neo. Short hairpin RNA vector C1, C2, C3, which contain σC gene, and shRNA vector NS1, NS2, NS3, which contain aNS gene, were constructed separately. The constructed shRNA vectors and negative control were co-transfected into DF-1 cells with the eukaryotic expression vector pEG- FP-σC and pEGFP-σNS, respectively. [ Result] Observation through fluorescence microscope indicated that the constructed 6 shRNA could inhibit the expression of fusion protein to different degrees. In addition, results of Real-time PCR suggested that C3 and NS1 have the best interference effect to the viral duplication in vitro. [ Conclusionl Construction and selection of specific shRNA expression vectors inhibiting Avian Reovirus are significant for researching effects of σC and oNS proteins in infection and duplication of ARV, providing new idea for ARV antiviral therapy.

Sequence and phylogenetic analysis of chicken reoviruses in China

supported by the China Animal Disease Prevention and Control Center;the China Agriculture Research System Poultry-Related Science and Technology Innovation Team of Peking, China （CARS-PSTP）

Temperature-regulated typeⅡgrass carp reovirus establishes latent infection in Ctenopharyngodon idella brain

Grass carp reovirus(GCRV)causes extensive infection and death in grass carp and black carp fingerlings,with a highly seasonal prevalence.Previous studies suggested that GCRV can become latent after primary infection.In this study,we investigated type II GCRV(GCRV-II)latency in asymptomatic grass carp with GCRV infection or exposure history.We found that during latent infection,GCRV-II was detectable only in the brain of grass carp,unlike the multi-tissue distribution observed in natural infection.GCRV-II only caused damage to the brain during latent infection,while in natural infection,brain,heart,and eye tissues had relatively higher viral loads.We also discovered viral inclusion bodies in infected fish brains.Additionally,GCRV-II distribution in grass carp was notably affected by ambient temperature,with the virus targeting the brain only during low temperatures and multi-tissue distribution during high temperatures.This study provides insights into the mechanisms of GCRV-II latent infection and reactivation and contributes to the prevention and control of GCRV pandemics.

Asymmetric reconstruction of the aquareovirus core at near-atomic resolution and mechanism of transcription initiation

Dear Editor,Aquareovirus(ARV,Reoviridae)causes hemorrhagic disease in the economically important golden shiner and grass carp of America and China,respectively(Nason et al.,2000;Fang et al.,2005).Reoviridae members are characterized by endogenous transcription of their multipartite genomes within capsids of 1-3 layers and are further classified based on the presence(Spinareovirinae subfamily,9 genera)or absence(Sedoreovirinae subfamily,6 genera)of mRNA-capping turrets along the innermost layer(King et al.,2012).

3D reconstruction and capsid protein characterization of grass carp reovirus

Grass carp reovirus (GCRV) is a relatively new virus first isolated in China and is a member of the Aquareovirus genus of the Reoviridae family. Recent report of genomic se-quencing showed that GCRV shared high degree of homology with mammalian reovirus (MRV). As a step of our effort to understand the structural basis of GCRV pathogenesis, we determined the three-dimensional (3D) structure of GCRV capsid at 17 ? resolution by electron cryomicro-scopy. Each GCRV capsid has a multilayered organization, consisting of an RNA core, an inner, middle and outer protein layer. The outer layer is made up of 200 trimers that are arranged on an incomplete T=13 icosahedral lattice. A characteristic feature of this layer is the depression re-sulting from the absence of trimers around the peripentonal positions, revealing the underlying trimers on the middle layer. There are 120 subunits in the inner layer arranged with T=1 symme-try. These structural features are common to other members of the Reoviridae. Moreover, SDS-PAGE analysis showed that GCRV virions contain seven structural proteins (VP1-VP7). These structural proteins have a high degree of sequence homology to MRV, consistent with the structural similarities observed in our study. The high structural similarities of isolated GCRV and MRV suggest that future structural studies focusing on GCRV entering into and replicating within its host cell are necessary in order to fully understand the structural basis of GCRV pathogenesis.

Antigenic analysis of grass carp reovirus using single-chain variable fragment antibody against IgM from Ctenopharyngodon idella

Grass carp （Ctenopharyngodon idella） is an important species of freshwater aquaculture fish in China. However, grass carp reovirus （GCRV） can cause fatal hemorrhagic disease in yearling populations. Until now, a strategy to define the antigenic ca- pacity of the virus＇s structural proteins for preparing an effective vaccine has not been available. In this study, some sin- gle-chain variable fragment antibodies （scFv）, which could specifically recognize grass carp IgM, were selected from a con- structed mouse naive antibody phage display cDNA library. The identified scFv C1B3 clone was shown to possess relatively higher specific binding activity to grass carp IgM. Furthermore, ELISA analysis indicated that the IgM level in serum from vi- rus-infected grass carp was more than two times higher than that of the control group at 5-7 days post infection. Moreover, Western blot analysis demonstrated that the outer capsid protein VP7 has a specific immuno-binding-reaction with the serum IgM from virus-infected grass carp. Our results suggest that VP7 can induce a stronger immune response in grass carp than the other GCRV structural proteins, which implies that VP7 protein could be used as a preferred immunogen for vaccine design.

Characterization of viral entry and infection of quantum dot-labeled grass carp reovirus

Dear Editor,Reoviruses are non-enveloped icosahedral virions with an outer capsid surrounding their cores,which harbor the10–12 segmented double-stranded RNA(ds RNA)genome.To date,there are 15 proposed genera in the family Reoviridae(King et al.,2012),including Aquareovirus.Generally,aquareoviruses are of low pathogenicity in aquaculture.However,grass carp reovirus(GCRV)is

呼肠孤病毒在肿瘤治疗研究进展

在过去的几十年间,溶瘤病毒（oncolytic virus,OV）在肿瘤治疗中已取得了很多突破性进展。野生型或经改造的OV可通过在肿瘤细胞内大量复制增殖来溶破肿瘤细胞,而对正常细胞无破坏作用。野生型呼肠孤病毒（reovirus）可选择性在肿瘤细胞内增殖,已证实其对多种来源的肿瘤细胞均可发挥溶瘤效应。