C6/36 Aedes albopictus cells have a dysfunctional antiviral RNA interference response

Mosquitoes rely on RNA interference (RNAi) as their primary defense against viral infections. To this end, the combination of RNAi and invertebrate cell culture systems has become an invaluable tool in studying virus-vector interactions. Nevertheless, a recent study failed to detect an active RNAi response to West Nile virus (WNV) infection in C6/36 (Aedes albopictus) cells, a mosquito cell line frequently used to study arthropod-borne viruses (arboviruses). Therefore, we sought to determine if WNV actively evades the host's RNAi response or if C6/36 cells have a dysfunctional RNAi pathway. C6/36 and Drosophila melanogaster S2 cells were infected with WNV (Flaviviridae), Sindbis virus (SINV, Togaviridae) and La Crosse virus (LACV, Bunyaviridae) and total RNA recovered from cell lysates. Small RNA (sRNA) libraries were constructed and subjected to high-throughput sequencing. In S2 cells, virus-derived small interfering RNAs (viRNAs) from all three viruses were predominantly 21 nt in length, a hallmark of the RNAi pathway. However, in C6/36 cells, viRNAs were primarily 17 nt in length from WNV infected cells and 26-27 nt in length in SINV and LACV infected cells. Furthermore, the origin (positive or negative viral strand) and distribution (position along viral genome) of S2 cell generated viRNA populations was consistent with previously published studies, but the profile of sRNAs isolated from C6/36 cells was altered. In total, these results suggest that C6/36 cells lack a functional antiviral RNAi response. These findings are analogous to the type-I interferon deficiency described in Vero (African green monkey kidney) cells and suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions at the molecular level.     

A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response

Invertebrate RNA viruses are targets of the host RNA interference (RNAi) pathway, which limits virus infection by degrading viral RNA substrates. Several insect RNA viruses encode suppressor proteins to counteract this antiviral response. We recently demonstrated that the dsDNA virus Invertebrate iridescent virus 6 (IIV-6) induces an RNAi response in Drosophila. Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R. Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs). We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex. Finally, we show that 340R is able to rescue a Flock House virus replicon that lacks its viral suppressor of RNAi. Together, our findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response.     

Novel Virus Discovery and Genome Reconstruction from Field RNA Samples Reveals Highly Divergent Viruses in Dipteran Hosts

We investigated whether small RNA (sRNA) sequenced from field-collected mosquitoes and chironomids (Diptera) can be used as a proxy signature of viral prevalence within a range of species and viral groups, using sRNAs sequenced from wild-caught specimens, to inform total RNA deep sequencing of samples of particular interest. Using this strategy, we sequenced from adult Anopheles maculipennis s.l. mosquitoes the apparently nearly complete genome of one previously undescribed virus related to chronic bee paralysis virus, and, from a pool of Ochlerotatus caspius and Oc. detritus mosquitoes, a nearly complete entomobirnavirus genome. We also reconstructed long sequences (1503-6557 nt) related to at least nine other viruses. Crucially, several of the sequences detected were reconstructed from host organisms highly divergent from those in which related viruses have been previously isolated or discovered. It is clear that viral transmission and maintenance cycles in nature are likely to be significantly more complex and taxonomically diverse than previously expected.     

Under siege: virus control in plant meristems and progeny

In the arms race between plants and viruses, two frontiers have been utilized for decades to combat viral infections in agriculture. First, many pathogenic viruses are excluded from plant meristems, which allows the regeneration of virus-free plant material by tissue culture. Second, vertical transmission of viruses to the host progeny is often inefficient, thereby reducing the danger of viral transmission through seeds. Numerous reports point to the existence of tightly linked meristematic and transgenerational antiviral barriers that remain poorly understood. In this review, we summarize the current understanding of the molecular mechanisms that exclude viruses from plant stem cells and progeny. We also discuss the evidence connecting viral invasion of meristematic cells and the ability of plants to recover from acute infections. Research spanning decades performed on a variety of virus/host combinations has made clear that, beside morphological barriers, RNA interference (RNAi) plays a crucial role in preventing—or allowing—meristem invasion and vertical transmission. How a virus interacts with plant RNAi pathways in the meristem has profound effects on its symptomatology, persistence, replication rates, and, ultimately, entry into the host progeny.

We review what is known about the biological mechanisms regulating virus exclusion from—or invasion of—plant host meristems and progeny, including possible consequences and implications of these phenomena.     

Hairpin RNA-mediated strategies for silencing of tomato leaf curl virus AC1 and AC4 genes for effective resistance in plants

RNA interference (RNAi) using short interfering RNAs (siRNAs) has been widely explored for the suppression of intracellular viral target mRNAs. On the basis of our previous work with stable silencing of Tomato leaf curl virus, in vivo by the antisense replicase gene (AC1) of the virus and characterizing AC4, as a small RNA regulator, besides its role in pathogenicity, we used four different plasmid vector-based siRNA generation strategies to silence viral genes (AC1 and AC4) of tomato leaf curl viruses. The RNAi target sequence were chosen from DNA A of the Tomato leaf curl virus (ToLCV) on the basis of conserved regions in AC1 with an overlapping sequences of the AC4 gene. Different hairpin RNA-mediated strategies like antisense, self-complementary inverted repeats, intron-spliced hairpin RNAs, and small hairpin RNAs were deployed for efficient and predictable resistance to the viruses. Here we present that appropriately designed siRNAs not only prevents RNAi suppression but also help in developing trait-stable transgenics. These strategies imply that ToLCV rep-driven RNAi, targeting AC4 and conserved viral sequences, provides a promising approach to suppress a wide spectrum ToLCV infection in the tomato.     

Novel Drosophila Viruses Encode Host-Specific Suppressors of RNAi

The ongoing conflict between viruses and their hosts can drive the co-evolution between host immune genes and viral suppressors of immunity. It has been suggested that an evolutionary ‘arms race’ may occur between rapidly evolving components of the antiviral RNAi pathway of Drosophila and viral genes that antagonize it. We have recently shown that viral protein 1 (VP1) of Drosophila melanogaster Nora virus (DmelNV) suppresses Argonaute-2 (AGO2)-mediated target RNA cleavage (slicer activity) to antagonize antiviral RNAi. Here we show that viral AGO2 antagonists of divergent Nora-like viruses can have host specific activities. We have identified novel Nora-like viruses in wild-caught populations of D. immigrans (DimmNV) and D. subobscura (DsubNV) that are 36% and 26% divergent from DmelNV at the amino acid level. We show that DimmNV and DsubNV VP1 are unable to suppress RNAi in D. melanogaster S2 cells, whereas DmelNV VP1 potently suppresses RNAi in this host species. Moreover, we show that the RNAi suppressor activity of DimmNV VP1 is restricted to its natural host species, D. immigrans. Specifically, we find that DimmNV VP1 interacts with D. immigrans AGO2, but not with D. melanogaster AGO2, and that it suppresses slicer activity in embryo lysates from D. immigrans, but not in lysates from D. melanogaster. This species-specific interaction is reflected in the ability of DimmNV VP1 to enhance RNA production by a recombinant Sindbis virus in a host-specific manner. Our results emphasize the importance of analyzing viral RNAi suppressor activity in the relevant host species. We suggest that rapid co-evolution between RNA viruses and their hosts may result in host species-specific activities of RNAi suppressor proteins, and therefore that viral RNAi suppressors could be host-specificity factors.     

RNA interference-mediated inhibition of Semliki Forest virus replication in mammalian cells

RNA interference (RNAi) has recently shown promise as a mode of inhibition of slowly replicating viruses causing chronic diseases such as hepatitis C. To investigate whether RNAi is also feasible for rapidly growing RNA viruses such as alphaviruses, we tested the ability of expressed short hairpin RNAs (shRNAs) to inhibit the Semliki Forest virus (SFV), a rapidly replicating positive-strand RNA virus. Plasmids expressing shRNAs targeting SFV target sequences under the control of a human U6 promoter were introduced into BHK-21 cells. The targets included sequences encoding nonstructural (nsP1, 2, and 4) and structural (capsid) proteins as well as nonviral sequences serving as control targets. Twenty-four to 48 hours following transfection with shRNA plasmids, the cells were infected with replication-competent or replication-deficient recombinant SFV expressing green fluorescent protein (GFP) at a multiplicity of infection (MOI) of approximately 5. Viral replication was monitored by fluorescence microscopy and flow cytometry. Specific and marked reduction of viral replication was observed with shRNAs targeting nsP1 and nsP4. The degree of inhibition of the replication-deficient SFV was >or=70% over a 5-day period, a level similar to the transfection efficiency, suggesting complete inhibition of nonreplicating virus in the transfected cell population. However, only nsP1 shRNA was inhibitory against replication-competent SFV (approximately 30%-50% reduction), and this effect was transient. No inhibition was observed with control shRNAs. In contrast to the recent success of RNAi approaches for slowly growing viruses, these results illustrate the challenge of inhibiting very rapidly replicating RNA viruses by RNAi. However, the addition of RNAi approaches to other antiviral modalities might improve the response to acute infections.     

MicroRNAs and human retroviruses

MicroRNAs (miRNAs) are small non-coding RNAs that control a multitude of critical processes in mammalian cells. Increasing evidence has emerged that host miRNAs serve in animal cells to restrict viral infections. In turn, many viruses encode RNA silencing suppressors (RSS) which are employed to moderate the potency of the cell's miRNA selection against viral replication. Some viruses also encode viral miRNAs. In this review, we summarize findings from human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) that illustrate examples of host cell miRNAs that target the viruses, of RSS encoded by viruses, and of host cell miRNA profile changes that are seen in infected cells. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.     

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system ¹. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance ^2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication ⁵. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses ^{4,6,7,8, 9}. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.     

Comparison of Viromes in Ticks from Different Domestic Animals in China

Ticks are involved in the transmission of various arboviruses and some tick-borne viruses pose significant threats to the health of humans or livestock. This study aimed to investigate the geographical distribution of tick species and tickassociated viruses in central and eastern China. Total 573 ticks from domestic animals including dogs, sheep and cattle were collected in 2017. Two genera of ticks were identified including Rhipicephalus and Haemaphysalis. Sequencing was performed on Miseq Illumina platform to characterize the tick viromes from the four different sampling locations.Following trimming, 13,640 reads were obtained and annotated to 19 virus families. From these sequences, above 37.74% of the viral reads were related to the RNA viruses. Virome comparison study revealed that the tick viral diversity was considerably different in the two identified tick genera. The viral diversity of R. microplus was significantly different from that of other Rhipicephalus species. On the other hand, substantial overlap in viral species was observed between the same genera. In addition, we found no evidence that the natural host played a major role in shaping virus diversity based on the comparison of their viromes. Rather, the geographic location seems to significantly influence the viral families. Phylogenetic study indicated that the novel negative-sense RNA viruses identified in this study was closely related to Bole tick virus 1 and 3 viruses. In conclusion, the present study provides a baseline for comparing viruses detected in ticks, according to species, natural hosts, and geographic locations.     

Simultaneous identification of DNA and RNA viruses present in pig faeces using process-controlled deep sequencing

Background

Animal faeces comprise a community of many different microorganisms including bacteria and viruses. Only scarce information is available about the diversity of viruses present in the faeces of pigs. Here we describe a protocol, which was optimized for the purification of the total fraction of viral particles from pig faeces. The genomes of the purified DNA and RNA viruses were simultaneously amplified by PCR and subjected to deep sequencing followed by bioinformatic analyses. The efficiency of the method was monitored using a process control consisting of three bacteriophages (T4, M13 and MS2) with different morphology and genome types. Defined amounts of the bacteriophages were added to the sample and their abundance was assessed by quantitative PCR during the preparation procedure.

Results

The procedure was applied to a pooled faecal sample of five pigs. From this sample, 69,613 sequence reads were generated. All of the added bacteriophages were identified by sequence analysis of the reads. In total, 7.7% of the reads showed significant sequence identities with published viral sequences. They mainly originated from bacteriophages (73.9%) and mammalian viruses (23.9%); 0.8% of the sequences showed identities to plant viruses. The most abundant detected porcine viruses were kobuvirus, rotavirus C, astrovirus, enterovirus B, sapovirus and picobirnavirus. In addition, sequences with identities to the chimpanzee stool-associated circular ssDNA virus were identified. Whole genome analysis indicates that this virus, tentatively designated as pig stool-associated circular ssDNA virus (PigSCV), represents a novel pig virus.

Conclusion

The established protocol enables the simultaneous detection of DNA and RNA viruses in pig faeces including the identification of so far unknown viruses. It may be applied in studies investigating aetiology, epidemiology and ecology of diseases. The implemented process control serves as quality control, ensures comparability of the method and may be used for further method optimization.     

RNA interference-mediated virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus

Several arenaviruses, including Lassa fever virus, cause severe, often lethal hemorrhagic fever in humans. No licensed vaccines are available in the United States, and currently there is no efficacious therapy to treat this viral infection. Therefore the importance of developing effective antiviral approaches to combat pathogenic arenaviruses is clear. Moreover, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important model for the study of viral persistence and associated diseases, as well as for exploring therapies to treat viral chronic infections. The use of small interfering RNAs (siRNAs) to downregulate gene expression via RNA interference (RNAi) has emerged as a powerful genetic tool for the study of gene function. In addition, the successful use of siRNAs to target a variety of animal viruses has led us to consider RNAi as a potential novel antiviral strategy. We have investigated the use of RNAi therapy against LCMV. Here, we show that siRNAs targeting sequences within the viral L polymerase and Z mRNAs inhibit LCMV multiplication in cultured cells. Unexpectedly, the antiviral efficacy of RNAi-based therapy against LCMV was highly dependent on the method used to deliver effector siRNA molecules. Thus, transfection of chemically synthesized siRNA pools to L and Z was ineffective in preventing virus multiplication. In contrast, targeting of the same viral L and Z gene products with siRNAs produced inside cells using a replication-deficient recombinant adenovirus expression system inhibited LCMV multiplication very efficiently. Notably, transduction with the replication-deficient recombinant adenovirus expression system to Z and L effectively cured persistently LCMV-infected cells, suggesting the feasibility of using RNAi therapy to combat viral chronic infections by riboviruses.     

ZWA: Viral genome assembly and characterization hindrances from virus-host chimeric reads; a refining approach

Viral metagenomics, also known as virome studies, have yielded an unprecedented number of novel sequences, essential in recognizing and characterizing the etiological agent and the origin of emerging infectious diseases. Several tools and pipelines have been developed, to date, for the identification and assembly of viral genomes. Assembly pipelines often result in viral genomes contaminated with host genetic material, some of which are currently deposited into public databases. In the current report, we present a group of deposited sequences that encompass ribosomal RNA (rRNA) contamination. We highlight the detrimental role of chimeric next generation sequencing reads, between host rRNA sequences and viral sequences, in virus genome assembly and we present the hindrances these reads may pose to current methodologies. We have further developed a refining pipeline, the Zero Waste Algorithm (ZWA) that assists in the assembly of low abundance viral genomes. ZWA performs context-depended trimming of chimeric reads, precisely removing their rRNA moiety. These, otherwise discarded, reads were fed to the assembly pipeline and assisted in the construction of larger and cleaner contigs making a substantial impact on current assembly methodologies. ZWA pipeline may significantly enhance virus genome assembly from low abundance samples and virus metagenomics approaches in which a small number of reads determine genome quality and integrity.     

Developing an effective RNA interference strategy against a plus-strand RNA virus: silencing of coxsackievirus B3 and its cognate coxsackievirus-adenovirus receptor

Coxsackievirus B3 (CVB-3) is a plus-strand RNA virus that is believed to be the most common causal agent of viral myocarditis. Since no specific treatment for CVB-3 infections is available to date, we and others have recently started to develop RNA interference (RNAi) approaches to prevent virus propagation. Here we describe our strategy for the development of efficient small interfering RNAs (siRNAs) against viral genomes. Initially, fusion constructs of a reporter (green fluorescent protein) and viral subgenomic fragments were employed to select active siRNAs against the virus. Moreover, in an attempt to achieve sustained virus silencing and reduce the risk of generating escape mutants, only highly efficient siRNAs directed against regions of the viral genome that are unlikely to tolerate mutations were considered for virus inhibition. Two siRNAs directed against the 3D RNA-dependent RNA polymerase were found to inhibit virus propagation by 80-90%. The protective effect of the efficient siRNAs lasted for several days. Furthermore, we have first evidence that inhibition of the cellular coxsackievirus-adenovirus receptor (CAR) by RNAi also reduces the virus titre. Our strategy is likely to be applicable to other (RNA) viruses as well.     

Virome analyses of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> using small RNA deep sequencing and PCR techniques reveal the presence of a potential new virus

Background

Hevea brasiliensis is an important commercial crop due to the high quality of the latex it produces; however, little is known about viral infections in this plant. The only virus described to infect H. brasiliensis until now is a Carlavirus, which was described more than 30?years ago. Virus-derived small interfering RNA (vsiRNAs) are the product of the plant’s antiviral defense triggered by dsRNA viral intermediates generated, during the replication cycle. These vsiRNAs are complementar to viral genomes and have been widely used to identify and characterize viruses in plants.

Methods

In the present study, we investigated the virome of leaf and sapwood samples from native H. brasiliensis trees collected in two geographic areas in the Brazilian Amazon. Small RNA (sRNA) deep sequencing and bioinformatic tools were used to assembly, identify and characterize viral contigs. Subsequently, PCR amplification techniques were performed to experimentally verify the presence of the viral sequences. Finally, the phylogenetic relationship of the putative new virus with related viral genomes was analyzed.

Results

Our strategy allowed the identification of 32 contigs with high similarity to viral reference genomes, from which 23 exhibited homology to viruses of the Tymoviridae family. The reads showed a predominant size distribution at 21?nt derived from both strands, which was consistent with the vsiRNAs profile. The presence and genome position of the viral contigs were experimentally confirmed using droplet digital PCR amplifications. A 1913 aa long fragment was obtained and used to infer the phylogenetic relationship of the putative new virus, which indicated that it is taxonomically related to the Grapevine fleck virus, genus Maculavirus. The putative new virus was named Hevea brasiliensis virus (HBrV) in reference to its host.

Conclusion

The methodological strategy applied here proved to be efficient in detecting and confirming the presence of new viral sequences on a ‘very difficult to manage’ sample. This is the second time that viral sequences, that could be ascribed as a putative novel virus, associated to the rubber tree has been identified.