Double-stranded RNA Segments Research Articles

Recovery of Recombinant Rotavirus Expressing Fluorescent Reporter Protein

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in infants and young children. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral ORFs, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.

English

Bluetongue virus (BTV) is the ‘type’ species of the genus Orbivirus within the family Reoviridae. The BTV genome is composed of ten linear segments of double-stranded RNA (dsRNA), each of which codes for at least one of ten distinct viral proteins. Phylogenetic analysis based on VP2, VP3, VP5, VP7 and NS3 gene has been advocated by different researchers around the world. However, very little information about the phylogenetic analysis based on the NS1 gene of BTV-16 isolates is available. In this study, a partial sequence of segment 5 (Seg 5) from the BTV-16 Hisar isolate was sequenced. Sequence analysis of the Seg 5 of the BTV-16 Hisar isolate revealed highest nucleotide sequence identity of 98.9% (BTV-21), -96.4% (BTV-9) with sequences of NS1 genes of Indian isolates and 97.8% (BTV-3),-93.4%(BTV-1) isolates from other countries. The lowest sequence identity detected was 91.2% between sequences of NS1 gene of JQ924824 of BTV 16 Indian isolate and JX861502 BTV-1 of France. The study also assessed the comparative identity from the deduced amino acid sequences and found maximum homology (100% identity) with Indian isolates and 95.2 to 98.8% identity with Western isolates with only three amino acid difference. Phylogenetic analysis of the NS1 gene of the BTV-16 Hisar isolate with others around the world has revealed two major clusters which includes the Indian (Eastern) linage and Western linage separately. The BTV-16 Hisar isolate was found to be closely related with the BTV-9 and 21 Indian isolates based on its NS1 gene. Phylogenetic analysis of segment 5 is highly conserved among the different serotypes however, NS1 of BTV-3 of USA clustered with the Eastern lineage indicating introduction of Western BTV strains and a possible reassortment between Eastern and Western field strains in India. Key words: Bluetongue virus, genome, NS1, Orbivirus, phylogeneticanalysis, segment-5, serotype.

Complete genome sequence of a novel mitovirus from the ectomycorrhizal fungus Geopora sumneriana.

A double-stranded RNA (dsRNA) segment was extracted from the ectomycorrhizal fungus Geopora sumneriana (Cooke) M. Torre, and its full-length cDNA sequence, comprising 3146 nucleotides, was determined. Sequence analysis revealed the presence of a large open reading frame (ORF) on the positive strand of this dsRNA segment when the mold mitochondrial genetic code was applied. The ORF encodes a putative RNA-dependent RNA polymerase (RdRp), which shares the highest degree of similarity with Tuber excavatum mitovirus, with 37.52% identity. This dsRNA segment represents the genome replication intermediate of a novel mitovirus that was tentatively designated as "Geopora sumneriana mitovirus 1" (GsMV1). Phylogenetic analysis further suggested that GsMV1 is a member of the family Narnaviridae. This is the first study reporting on a mitovirus genome sequence in the ectomycorrhizal fungus G. sumneriana.

Complete genome sequence and phylogenetic analysis of a novel aquareovirus isolated from a diseased marbled eel (Anguilla marmorata).

Marbled eel reovirus (MERV) is an aquareovirus (AQRV) isolated from diseased marbled eels (Anguilla marmorata) with petechial skin hemorrhage. In this study, we propagated MERV in a cell line derived from the brain of Aequidens rivulatus and purified viral particles by using a discontinuous cesium chloride gradient. Genomic RNA sequences were obtained through next-generation sequencing. MERV, similar to most other AQRVs, showed the presence of 11 double-stranded RNA segments encoding 12 proteins; however, the genome sequence displayed very little similarity to known AQRV sequences. Furthermore, the structural proteins of MERV were most closely related to American grass carp reovirus with sequence identity values of no more than 64.89%. Phylogenetic analysis based on the sequences of structural proteins indicated that MERV shows an evolutionary history between AQRV-B and -G, which belong to the saline and freshwater environment subgroups, respectively. We also observed that MERV showed a closer relationship to orthoreoviruses based on the protein sequences of NS38 and NS73. In summary, MERV is a novel AQRV that could be classified as a member of the new proposed AQRV species "Aquareovirus H". The taxonomic assignments and evolution of AQRVs thus warrant further investigation.

Abstract 2169: Doxorubicin conjugation to reovirus enhances tumor cell-directed oncolysis

Abstract Breast cancer remains the most diagnosed type of cancer and second leading cause of cancer-related deaths in women in the United States. Triple-negative breast cancer (TNBC) constitutes 10-20% of breast cancer cases. TNBCs are characterized by lack of estrogen (ER) and progesterone (PR) receptors and growth factor receptor HER2/Neu on the cell surface. Therapies that target ER, PR, or HER2 are thus ineffective against TNBC. TNBC is primarily treated with cytotoxic chemotherapy or radiation therapy, which can cause significant damage to healthy cells and tissue. Therefore, there is need for therapies that selectively kill TNBC cells with reduced off-tumor effects. The concept of oncolytic virotherapy has existed since the early 1900s. However, oncolytic viruses have limited efficacy as single agents. Mammalian orthoreovirus (reovirus) is a non-enveloped, segmented double-stranded RNA virus with tropism for transformed cells. Reovirus is in Phase I - III clinical trials to test its oncolytic efficacy against a variety of cancers. Success has been limited and few trials have tested reovirus against breast cancer. In a high-throughput screen of small molecule inhibitors, we identified the topoisomerase II inhibitor doxorubicin hydrochloride (dox) as an enhancer of reovirus infectivity in the TNBC cell line MDA-MB-231. To better control dox delivery and enhance reovirus oncolytic potential, we chemically conjugated dox to reovirus virions (reo-dox). While reo-dox attachment to MDA-MB-231 cells is slightly impaired, viral replication is largely unaffected. Infection of MDA-MB-231 cells with reo-dox resulted in increased cytopathicity and faster induction of cell death than virus alone. MDA-MB-231 cells infected with reo-dox, but not virus alone, induced DNA double-strand breaks and activation of DNA damage response. Together, our findings show that reo-dox exhibits superior toxicity in TNBC cells than virus alone. Future studies will define the mechanism of enhanced cytopathicity of reo-dox and oncolytic efficacy of dox-conjugated reovirus in vivo. Delivery of small molecule inhibitors via conjugation to reovirus particles may provide an effective new method to directly target and kill cancer cells while minimizing toxicity to healthy cells and tissues. Citation Format: Jameson T. Berry, Bernardo A. Mainou. Doxorubicin conjugation to reovirus enhances tumor cell-directed oncolysis [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 2169.

Prevalence and Strains of Colorado Tick Fever Virus in Rocky Mountain Wood Ticks in the Bitterroot Valley, Montana.

The Rocky Mountain wood tick, Dermacentor andersoni, has long been known to transmit human pathogens. Within the Bitterroot Valley, Ravalli County, Montana, these agents include Rickettsia rickettsii, Francisella tularensis, and Colorado tick fever virus (CTFV). Found in the western United States where wood ticks occur, CTFV causes a biphasic, febrile illness in humans and persists in enzootic cycles involving the ticks and small mammals. CTFV belongs to the genus Coltivirus, family Reoviridae, whose genome consists of 12 double-stranded RNA segments. Previous studies revealed the presence of CTFV-infected ticks and rodents in select locations within the valley in the 1960s and 1970s, using animal and cell culture methods for detection. We aimed to determine the range and prevalence of the virus in adult questing ticks throughout the valley using molecular tools and to examine the genomic variation between virus strains. Adult D. andersoni ticks were collected during 2002-2003 and 2009-2013. RNA extractions and reverse transcription-polymerase chain reaction were performed on 921 ticks, of which 61 ticks were positive for CTFV, resulting in a 6.6% prevalence of infection. Four genetic loci, one from each of the segments 9, 10, 11, and 12, within the viral genome were sequenced. Reassortment was detected between CTFV sequence strains within the valley. This study confirmed the prevalence of CTFV in D. andersoni ticks within the Bitterroot Valley, which has remained at levels found in the 1950s and 60s. Additional CTFV sequences were obtained and evidence of reassortment was observed between strains within the valley.

Molecular typing of Bluetongue virus using the nCounter® analysis system platform

Bluetongue virus (BTV) is a segmented double-stranded RNA virus, existing in multiple serotypes, belonging to the genus Orbivirus of the family Reoviridae. BTV causes Bluetongue (BT), a major OIE-listed disease of ruminants. Identification of BTV serotype is accomplished using multiple typing assays and tends to be executed based on the known epidemiological situation within a given country. Samples containing multiple serotypes, particularly those containing novel introductions, may therefore be missed. The aim of this work was to optimize the nCounter® Analysis System Microarray platform (NanoString technologies), that would simultaneously identify all BTV serotypes and co-infections in analyzed samples. Probes were designed according to all Seg-2 sequences, coding for VP2 proteins which determine serotype specificity, available on line. A specific BTV CodeSet of probes was optimized. Experiments were performed with 30 BTV isolates and with 46 field samples previously shown to be infected with BTV by classical molecular assays. All BTV isolates were correctly identified and the expected BTV serotype was recognized in 35 field samples with CT values between 22.0–33.0. In turn, it was unable to identify 11 samples with CT values between 29.0–38.0. Although specificity of the assay needs to be further investigated against a larger panel of BTVs collected worldwide, RNA loads, which are normally detected in blood samples during the acute phase of infection, are within the range of CT values detectable by the BTV CodeSet. We propose the NanoString RNA microarray as a first-line molecular diagnostic tool for identification and typing of BTV. Once identification of the index cases is performed, diagnosis of the following samples may be performed by specific, more sensitive and cheaper PCR-based tools.

A Reverse Genetics System for Cypovirus Based on a Bacmid Expressing T7 RNA Polymerase.

Dendrolimus punctatus cypovirus (DpCPV), belonging to the genus Cypovirus within the family Reoviridae, is considered the most destructive pest of pine forests worldwide. DpCPV has a genome consisting of 10 linear double-stranded RNA segments. To establish a reverse genetics system, we cloned cDNAs encoding the 10 genomic segments of DpCPV into three reverse genetics vectors in which each segment was transcribed under the control of a T7 RNA polymerase promoter and terminator tagged with a hepatitis delta virus ribozyme sequence. We also constructed a vp80-knockout Autographa californica multiple nucleopolyhedrovirus bacmid to express a T7 RNA polymerase codon-optimized for Sf9 cells. Following transfection of Sf9 cells with the three vectors and the bacmid, occlusion bodies (OBs) with the typical morphology of cypovirus polyhedra were observed by optical microscopy. The rescue system was verified by incorporation of a HindIII restriction enzyme site null mutant of the 9th genomic segment. Furthermore, when we co-transfected Sf9 cells with the reverse genetics vectors, the bacmid, and an additional vector bearing an egfp gene flanked with the 5′ and 3′ untranslated regions of the 10th genomic segment, aggregated green fluorescence co-localizing with the OBs was observed. The rescued OBs were able to infect Spodopetra exigua larvae, although their infectivity was significantly lower than that of wild-type DpCPV. This reverse genetics system for DpCPV could be used to explore viral replication and pathogenesis and to facilitate the development of novel bio-insecticides and expression systems for exogenous proteins.

Molecular Characterization of a Debilitation-Associated Partitivirus Infecting the Pathogenic Fungus Aspergillus flavus.

The opportunistic human pathogenic fungus Aspergillus flavus is known to be infected with mycoviruses. In this study, we report a novel mycovirus A. flavus partitivirus 1 (AfPV1) that was originally isolated from the abnormal colonial morphology isolate LD-3-8 of A. flavus. AfPV1 has spherical virus-like particles about 40 nm in diameter, and three double-stranded RNA (dsRNA) segments (dsRNA1, 2, and 3 with lengths of 1.7, 1.4, and 1.1 kbp, respectively) were packaged in the virions. dsRNA1, dsRNA2, and dsRNA3 each contained a single open reading frame and potentially encoded 62, 42, and 32 kDa proteins, respectively. The dsRNA1 encoded protein shows similarity to the RNA-dependent RNA polymerase (RdRp) of partitiviruses, and the dsRNA2 product has no significant similarity to any other capsid protein (CP) in the GenBank databases, beside some homology with the hypothetical “capsid” protein of a few partitiviruses. The dsRNA3 encodes a protein with no similarity to any protein in the GenBank database. SDS-PAGE and polypeptide mass fingerprint-mass spectrum (PMF-MS) analyses indicated that the CP of the AfPV1 was encoded by dsRNA2. Phylogenetic analysis showed that the AfPV1 and relative viruses were found in an unclassified group inside the Partitiviridae family. AfPV1 seems to result in debilitation symptoms, but had no significant effects to murine pathogenicity. These findings provide new insights into the partitiviruses taxonomy and the interactions between viruses and A. flavus.

Function, Architecture, and Biogenesis of Reovirus Replication Neoorganelles.

Most viruses that replicate in the cytoplasm of host cells form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Reoviruses are common pathogens of mammals that have been linked to celiac disease and show promise for oncolytic applications. These viruses form nonenveloped, double-shelled virions that contain ten segments of double-stranded RNA. Replication organelles in reovirus-infected cells are nucleated by viral nonstructural proteins µNS and σNS. Both proteins partition the endoplasmic reticulum to form the matrix of these structures. The resultant membranous webs likely serve to anchor viral RNA–protein complexes for the replication of the reovirus genome and the assembly of progeny virions. Ongoing studies of reovirus replication organelles will advance our knowledge about the strategies used by viruses to commandeer host biosynthetic pathways and may expose new targets for therapeutic intervention against diverse families of pathogenic viruses.

Isolation and genomic characterization of a cypovirus from the oleander hawk moth, Daphnis nerii.

The oleander hawk moth, Daphnis nerii, is a serious pest of plants belonging to the family Apocynaceae. Thus far, pathogen infection has not been reported in D. nerii. In this study, a new cytoplasmic polyhedrosis virus (cypovirus; CPV) was isolated from naturally diseased D. nerii larvae and named DnCPV-23. Virions were observed in ultrathin sections of DnCPV polyhedral bodies. Electrophoretic analysis revealed that the DnCPV genome consisted of 10 segments of double-stranded RNA (dsRNA). cDNA copies of these dsRNA segments were amplified using the method of full-length amplification of cDNAs (FLAC), cloned, and sequenced. Sequencing results showed that all segments contained one open reading frame (ORF); They shared the conserved terminal sequences AGUCAAA and AGC at 5' and 3' ends respectively, except segment 4, which is different from previously reported 22 cypoviruses. Phylogenetic analysis based on amino acid sequences of polyhedrin (encoded by segment 10) indicated that this CPV was closely related to CPV type 19. Altogether, DnCPV-23 is a new type of cypovirus.

Enterovirus particles expel capsid pentamers to enable genome release

Viruses from the genus Enterovirus are important human pathogens. Receptor binding or exposure to acidic pH in endosomes converts enterovirus particles to an activated state that is required for genome release. However, the mechanism of enterovirus uncoating is not well understood. Here, we use cryo-electron microscopy to visualize virions of human echovirus 18 in the process of genome release. We discover that the exit of the RNA from the particle of echovirus 18 results in a loss of one, two, or three adjacent capsid-protein pentamers. The opening in the capsid, which is more than 120 Å in diameter, enables the release of the genome without the need to unwind its putative double-stranded RNA segments. We also detect capsids lacking pentamers during genome release from echovirus 30. Thus, our findings uncover a mechanism of enterovirus genome release that could become target for antiviral drugs.

A novel chrysovirus from a clinical isolate of Aspergillus thermomutatus affects sporulation.

A clinical isolate of Aspergillus thermomutatus (Teleomorph: Neosartorya pseudofischeri) was found to contain ~35 nm isometric virus-like particles associated with four double-stranded (ds) RNA segments, each of which coded for a single open reading frame. The longest dsRNA element (3589 nt) encodes a putative RNA-dependent RNA polymerase (1114 aa), the second longest dsRNA element (2772 nt) encodes a coat protein (825 aa), and the other two dsRNAs (2676 nt, 2514 nt) encode hypothetical proteins of 768 aa and 711 aa, respectively. Phylogenetic analysis of the amino acid sequences showed 41–60% similarity to the proteins coded by the dsRNAs of the most closely related virus, Penicillium janczewskii chrysovirus 2, indicating that it is a new species based on the International Committee on Taxonomy of Viruses criteria for the genus Chrysovirus. This is the first virus reported from A. thermomutatus and was tentatively named Aspergillus thermomutatus chrysovirus 1. A virus free line of the fungal isolate, cured by cycloheximide treatment, produced large numbers of conidia but no ascospores at both 20°C and 37°C, whereas the virus infected line produced ten-fold fewer conidia at 20°C and a large number of ascospores at both temperatures. The effects of the virus on fungal sporulation have interesting implications for the spread of the fungus and possible use of the virus as a biological control agent.

First report of a novel alphapartitivirus in the basidiomycete Rhizoctonia oryzae-sativae.

Rhizoctonia oryzae-sativae is a soil-borne basidiomycete fungus that causes aggregate sheath spot disease on rice worldwide. Here, we report the complete genome sequence of a partitivirus designated as Rhizoctonia oryzae-sativae partitivirus 1 (RosPV1) infecting this fungus. The genome of RosPV1 consists of two double-stranded RNA(dsRNA) segments. The larger segment, designated as dsRNA-1 (1,961 bp), contains a single open reading frame (ORF) that encodes a putative polypeptide with a conserved RNA-dependent RNA polymerase (RdRp) domain. The smaller segment, dsRNA-2 (1,819bp), also has a single ORF, which is predicted to encode the capsid protein (CP). BLAST searches and phylogenetic analyses suggested that RosPV1 is a representative member of a new species within the genus Alphapartitivirus. This is the first report of an alphapartitivirus infecting the fungus R. oryzae-sativae.

Fusarium oxysporum f. sp. dianthi virus 1 Accumulation Is Correlated with Changes in Virulence and Other Phenotypic Traits of Its Fungal Host.

Fusarium oxysporum f. sp. dianthi virus 1 (FodV1) was detected in isolate 116 (116V+) of Fusarium oxysporum f. sp. dianthi, reaching such a high accumulation level that it was clearly visible after agarose gel electrophoresis of total DNA extracts. FodV1 consists of four double-stranded RNA segments that correspond to a new mycovirus in the Chrysoviridae family. We obtained an isolate of F. oxysporum f. sp. dianthi 116 (116V-) with only a residual level of FodV1 RNA accumulation by single-conidia selection. Compared with 116V-, isolate 116V+ showed significant phenotypic alterations in vegetative growth and virulence. Thus, the presence of a high titer of mycovirus FodV1 was associated with a modified morphology and a reduced growth of the colonies on solid medium, and with a diminished conidiation in liquid medium. Inoculation of four susceptible carnation cultivars with either 116V- or 116V+ showed that the presence of a high titer of FodV1 was also correlated with a significantly reduced virulence of its fungal host. All of the results suggest that FodV1 could be associated with hypovirulence, identifying it as a potential biocontrol agent for Fusarium wilt of carnation. This is the first report of a mycovirus potentially associated with the induction of hypovirulence in the species F. oxysporum.

Novel Partitivirus Enhances Virulence of and Causes Aberrant Gene Expression in Talaromyces marneffei.

ABSTRACTTalaromyces marneffei is the most important thermal dimorphic fungus causing systemic mycosis in Southeast Asia. We report the discovery of a novel partitivirus, Talaromyces marneffei partitivirus-1 (TmPV1). TmPV1 was detected in 7 (12.7%) of 55 clinical T. marneffei isolates. Complete genome sequencing of the seven TmPV1 isolates revealed two double-stranded RNA (dsRNA) segments encoding RNA-dependent RNA polymerase (RdRp) and capsid protein, respectively. Phylogenetic analysis showed that TmPV1 occupied a distinct clade among the members of the genus Gammapartitivirus. Transmission electron microscopy confirmed the presence of isometric, nonenveloped viral particles of 30 to 45 nm in diameter, compatible with partitiviruses, in TmPV1-infected T. marneffei. Quantitative reverse transcription-PCR (qRT-PCR) demonstrated higher viral load of TmPV1 in the yeast phase than in the mycelial phase of T. marneffei. Two virus-free isolates, PM1 and PM41, were successfully infected by purified TmPV1 using protoplast transfection. Mice challenged with TmPV1-infected T. marneffei isolates showed significantly shortened survival time (P < 0.0001) and higher fungal burden in organs than mice challenged with isogenic TmPV1-free isolates. Transcriptomic analysis showed that TmPV1 causes aberrant expression of various genes in T. marneffei, with upregulation of potential virulence factors and suppression of RNA interference (RNAi)-related genes. This is the first report of a mycovirus in a thermally dimorphic fungus. Further studies are required to ascertain the mechanism whereby TmPV1 enhances the virulence of T. marneffei in mice and the potential role of RNAi-related genes in antiviral defense in T. marneffei.

Stability of local secondary structure determines selectivity of viral RNA chaperones.

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA–RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA–RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

Infectious bursal disease virus in Algeria: Detection of highly pathogenic reassortant viruses

Infectious bursal disease (IBD) is an immunosuppressive viral disease, present worldwide, which causes mortality and immunosuppression in young chickens. The causative agent, the Avibirnavirus IBDV, is a non-enveloped virus whose genome consists of two segments (A and B) of double-stranded RNA. Different pathotypes of IBDV exist, ranging from attenuated vaccine strains to very virulent viruses (vvIBDV). In Algeria, despite the prophylactic measures implemented, cases of IBD are still often diagnosed clinically and the current molecular epidemiology of IBDV remains unknown. The presence of the virus and especially of strains genetically close to vvIBDV was confirmed in 2000 by an unpublished OIE report. In this study, nineteen IBDV isolates were collected in Algeria between September 2014 and September 2015 during clinical outbreaks. These isolates were analyzed at the genetic, antigenic and pathogenic levels. Our results reveal a broad genetic and phenotypic diversity of pathogenic IBDV strains in Algeria, with, i) the circulation of viruses with both genome segments related to European vvIBDV, which proved as pathogenic for specific pathogen-free chickens as vvIBDV reference strain, ii) the circulation of viruses closely related - yet with a specific segment B - to European vvIBDV, their pathogenicity being lower than reference vvIBDV, iii) the detection of reassortant viruses whose segment A was related to vvIBDV whereas their segment B did not appear closely related to any reference sequence. Interestingly, the pathogenicity of these potentially reassortant strains was comparable to that of reference vvIBDV. All strains characterized in this study exhibited an antigenicity similar to the cognate reference IBDV strains. These data reveal the continuous genetic evolution of IBDV strains in Algerian poultry through reassortment and acquisition of genetic material of unidentified origin. Continuous surveillance of the situation as well as good vaccination practice associated with appropriate biosecurity measures are necessary for disease control.

Identification of a Small Molecule That Compromises the Structural Integrity of Viroplasms and Rotavirus Double-Layered Particles.

Despite the availability of two attenuated vaccines, rotavirus (RV) gastroenteritis remains an important cause of mortality among children in developing countries, causing about 215,000 infant deaths annually. Currently, there are no specific antiviral therapies available. RV is a nonenveloped virus with a segmented double-stranded RNA genome. Viral genome replication and assembly of transcriptionally active double-layered particles (DLPs) take place in cytoplasmic viral structures called viroplasms. In this study, we describe strong impairment of the early stages of RV replication induced by a small molecule known as an RNA polymerase III inhibitor, ML-60218 (ML). This compound was found to disrupt already assembled viroplasms and to hamper the formation of new ones without the need for de novo transcription of cellular RNAs. This phenotype was correlated with a reduction in accumulated viral proteins and newly made viral genome segments, disappearance of the hyperphosphorylated isoforms of the viroplasm-resident protein NSP5, and inhibition of infectious progeny virus production. In in vitro transcription assays with purified DLPs, ML showed dose-dependent inhibitory activity, indicating the viral nature of its target. ML was found to interfere with the formation of higher-order structures of VP6, the protein forming the DLP outer layer, without compromising its ability to trimerize. Electron microscopy of ML-treated DLPs showed dose-dependent structural damage. Our data suggest that interactions between VP6 trimers are essential, not only for DLP stability, but also for the structural integrity of viroplasms in infected cells.IMPORTANCE Rotavirus gastroenteritis is responsible for a large number of infant deaths in developing countries. Unfortunately, in the countries where effective vaccines are urgently needed, the efficacy of the available vaccines is particularly low. Therefore, the development of antivirals is an important goal, as they might complement the available vaccines or represent an alternative option. Moreover, they may be decisive in fighting the acute phase of infection. This work describes the inhibitory effect on rotavirus replication of a small molecule initially reported as an RNA polymerase III inhibitor. The molecule is the first chemical compound identified that is able to disrupt viroplasms, the viral replication machinery, and to compromise the stability of DLPs by targeting the viral protein VP6. This molecule thus represents a starting point in the development of more potent and less cytotoxic compounds against rotavirus infection.

A plasmid-based reverse genetics system for rotaviruses

Rotavirus (RV), a non-enveloped icosahedral virus containing eleven gene segments of double-stranded RNA, is the leading cause of severe, acute diarrhea among infants and young children worldwide. Safe and effective rotavirus vaccines have been available since 2006, and have markedly reduced the number of deaths by severe gastroenteritis. However, rotaviruses are still responsible for approximately 200,000 deaths annually worldwide. Reverse genetics systems for the manipulation of viral genomes are a powerful approach for studying viral replication and pathogenesis, and for developing vaccines and viral vectors. The understanding of the molecular mechanisms underlying RV pathogenesis, or development of next generation vaccines, has been hampered by the lack of a complete reverse genetics system. Recently, we developed a novel reverse genetics system which enabled recovery of recombinant RVs entirely from cloned cDNAs. This new strategy requires co-expression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. In this review, the strategies and history of the development of reverse genetics systems for the family Reoviridae are described.