RNA virome abundance and diversity is associated with host age in a bird species.

Despite the ongoing interest in virus discovery, little is known about the factors that shape communities of viruses within individual hosts. Here, we address how virus communities might be impacted by the age of the hosts they infect, using total RNA sequencing to reveal the RNA viromes of different age groups of Ruddy Turnstones (Arenaria interpres). From oropharyngeal and cloacal swabs we identified 14 viruses likely infecting birds, 11 of which were novel, including members of the Reoviridae, Astroviridae, and Picornaviridae. Strikingly, 12 viruses identified were from juvenile birds sampled in the first year of their life, compared to only two viruses in adult birds. Both viral abundance and alpha diversity were marginally higher in juvenile than adult birds. As well as informing studies of virus ecology, that host age might be associated with viral composition is an important consideration for the future surveillance of novel and emerging viruses.

Ginsenoside Rg3 inhibits grass carp reovirus replication in grass carp ovarian epithelial cells.

Ginseng exhibits multiple medicinal properties, including the improvement of immune function and enhancing disease resistance. In this study, we investigated the inhibitory effects of ginsenoside Rg3 on grass carp reovirus (GCRV) infection of grass carp ovarian (CO) epithelial cells, in order to provide a baseline framework for future high-efficacy antiviral drug screening investigations. Ginsenoside Rg3 was added to GCRV-infected CO cells, and cells were cultured at 27 °C before cell proliferation was measured by MTT assays. Label-free real-time cellular analysis (RTCA) after 72 h of experimentation demonstrated that 100 µg/mL ginsenoside Rg3 treatment had the highest inhibitory effect on GCRV (among 1,10,100 µg/mL treatments). We then measured the capacity for cellular antioxidant ability. Cells treated with 1,10,100 µg/mL ginsenoside Rg3 exhibited increases in Total Antioxidant Capacity activity relative to controls, respectively. Furthermore, Antioxidant assay and reverse transcript quantitative polymerase chain reaction (RT-qPCR) showed that ginsenoside Rg3 were efficient to restrain the replication of GCRV in CO cells. Expression analysis of immune-related genes via RT-qPCR showed that treatment with ginsenoside Rg3 promoted expression of IRF-3 and IRF-7 increases, respectively. Moreover, expression of IFN-1 was induced, which then inhibition the expression of tumor necrosis factor-alpha (TNF-α). In conclusion, we demonstrated that ginsenoside Rg3 promotes CO cell proliferation, inhibits GCRV activity, promotes CO cell immune activities, and thereby enhances the resistance of CO to GCRV infection.

Enteric viruses exploit the microbiota to promote infection.

Enteric viruses infect the mammalian gastrointestinal tract which is home to a diverse community of intestinal bacteria. Accumulating evidence suggests that certain enteric viruses utilize these bacteria to promote infection. While this is not surprising considering their proximity, multiple viruses from different viral families have been shown to bind directly to bacteria or bacterial components to aid in viral replication, pathogenesis, and transmission. These data suggest that the concept of a single virus infecting a single cell, independent of the environment, needs to be reevaluated. In this review, I will discuss the current knowledge of enteric virus-bacterial interactions and discuss the implications for viral pathogenesis and transmission.

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.

Inhibitor analysis revealed that clathrin-mediated endocytosis is involed in cellular entry of type III grass carp reovirus.

BACKGROUND: Grass carp (Ctenopharyngodon idella) hemorrhagic disease is caused by an acute infection with grass carp reovirus (GCRV). The frequent outbreaks of this disease have suppressed development of the grass carp farming industry. GCRV104, the representative strain of genotype III grass carp (Ctenopharyngodon idella) reovirus, belongs to the Spinareovirinae subfamily and serves as a model for studying the strain of GCRV which encodes an outer-fiber protein. There is no commercially available vaccine for this genotype of GCRV. Therefore, the discovery of new inhibitors for genotype III of GCRV will be clinically beneficial. In addition, the mechanism of GCRV with fiber entry into cells remains poorly understood. METHODS: Viral entry was determined by a combination of specific pharmacological inhibitors, transmission electron microscopy, and real-time quantitative PCR. RESULTS: Our results demonstrate that both GCRV-JX01 (genotype I) and GCRV104 (genotype III) of GCRV propagated in the grass carp kidney cell line (CIK) with a typical cytopathic effect (CPE). However, GCRV104 replicated slower than GCRV-JX01 in CIK cells. The titer of GCRV-JX01 was 1000 times higher than GCRV104 at 24 h post-infection. We reveal that ammonium chloride, dynasore, pistop2, chlorpromazine, and rottlerin inhibit viral entrance and infection, but not nystatin, methyl-ß-cyclodextrin, IPA-3, amiloride, bafilomycin A1, nocodazole, and latrunculin B. Furthermore, GCRV104 and GCRV-JX01 infection of CIK cells depended on dynamin and the acidification of the endosome. This was evident by the significant inhibition following prophylactic treatment with the lysosomotropic drug ammonium chloride or dynasore. CONCLUSIONS: Taken together, our data have suggested that GCRV104 enters CIK cells through clathrin-mediated endocytosis in a pH-dependent manner. We also suggest that dynamin is critical for efficient viral entry. Additionally, the phosphatidylinositol 3-kinase inhibitor wortmannin and the protein kinase C inhibitor rottlerin block GCRV104 cell entry and replication.

Isolation and characterization of the fall Chinook aquareovirus.

BACKGROUND: Salmon are paramount to the economy, ecology, and history of the Pacific Northwest. Viruses constitute one of the major threats to salmon health and well-being, with more than twenty known virus species that infect salmon. Here, we describe the isolation and characterization of the fall Chinook aquareovirus, a divergent member of the species Aquareovirus B within the family Reoviridae. METHODS: The virus was first found in 2014 as part of a routine adult broodstock screening program in which kidney and spleen tissue samples from healthy-appearing, adult fall Chinook salmon (Oncorhynchus tshawytscha) returning to a hatchery in Washington State produced cytopathic effects when inoculated onto a Chinook salmon embryo cell line (CHSE-214). The virus was not able to be confirmed by an RT-PCR assay using existing aquareovirus pan-species primers, and instead was identified by metagenomic next-generation sequencing. Metagenomic next-generation sequencing was used to recover the full genome and completed using 3' RACE. RESULTS: The genome of the fall Chinook aquareovirus contains 11 segments of double-stranded RNA totaling 23.3 kb, with each segment flanked by the canonical sequence termini found in the aquareoviruses. Sequence comparisons and a phylogenetic analysis revealed a nucleotide identity of 63.2% in the VP7 gene with the Green River Chinook virus, placing the new isolate in the species Aquareovirus B. A qRT-PCR assay was developed targeting the VP2, which showed rapid growth of the isolate during the initial 5 days in culture using CHSE-214 cells. CONCLUSIONS: This sequence represents the first complete genome of an Aquareovirus B species. Future studies will be required to understand the potential pathogenicity and epidemiology of the fall Chinook aquareovirus.

A novel method for transmitting southern rice black-streaked dwarf virus to rice without insect vector.

BACKGROUND: Southern rice black-streaked dwarf virus (SRBSDV) has spread from the south of China to the north of Vietnam in the past few years, and has severely influenced rice production. However, previous study of traditional SRBSDV transmission method by the natural virus vector, the white-backed planthopper (WBPH, Sogatella furcifera), in the laboratory, researchers are frequently confronted with lack of enough viral samples due to the limited life span of infected vectors and rice plants and low virus acquisition and inoculation efficiency by the vector. Meanwhile, traditional mechanical inoculation of virus only apply to dicotyledon because of the higher content of lignin in the leaves of the monocot. Therefore, establishing an efficient and persistent-transmitting model, with a shorter virus transmission time and a higher virus transmission efficiency, for screening novel anti-SRBSDV drugs is an urgent need. METHODS: In this study, we firstly reported a novel method for transmitting SRBSDV in rice using the bud-cutting method. The transmission efficiency of SRBSDV in rice was investigated via the polymerase chain reaction (PCR) method and the replication of SRBSDV in rice was also investigated via the proteomics analysis. RESULTS: Rice infected with SRBSDV using the bud-cutting method exhibited similar symptoms to those infected by the WBPH, and the transmission efficiency (>80.00%), which was determined using the PCR method, and the virus transmission time (30 min) were superior to those achieved that transmitted by the WBPH. Proteomics analysis confirmed that SRBSDV P1, P2, P3, P4, P5-1, P5-2, P6, P8, P9-1, P9-2, and P10 proteins were present in infected rice seedlings infected via the bud-cutting method. CONCLUSION: The results showed that SRBSDV could be successfully transmitted via the bud-cutting method and plants infected SRBSDV exhibited the symptoms were similar to those transmitted by the WBPH. Therefore, the use of the bud-cutting method to generate a cheap, efficient, reliable supply of SRBSDV-infected rice seedlings should aid the development of disease control strategies. Meanwhile, this method also could provide a new idea for the other virus transmission in monocot.

Co-infection of two reoviruses increases both viruses accumulation in rice by up-regulating of viroplasm components and movement proteins bilaterally and RNA silencing suppressor unilaterally.

BACKGROUND: Synergism between southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) not only aggravates disease symptoms but also enhances their vector acquisition efficiencies by increasing both viruses' titers in co-infected rice plants, which may exacerbate the epidemic of both viruses and cause significant damage to rice production. The molecular mechanism of viral synergism of these two viruses remains unexplored. METHODS: Single and double infection of SRBSDV and RRSV were obtained with the viruliferous white-backed planthopper and brown planthopper inoculation on four-leaf stage rice seedlings, respectively, under experimental condition. The second upper leaf from each inoculated rice plants were collected at 9, 15, and 20 days post inoculation (dpi) and used for relative quantification of 13 SRBSDV genes and 11 RRSV genes by the reverse-transcription quantitative PCR. Viral gene expression levels were compared between singly and doubly infected samples at the same stage. RESULTS: The movement protein and viroplasm matrix-related genes as well as the structural (capsid) protein genes of both viruses were remarkably up-regulated at different time points in the co-infected rice plants compared with the samples singly infected with SRBSDV or RRSV, however, the RNA silencing suppressor (P6) of only RRSV, but not of both the viruses, was up-regulated. CONCLUSIONS: The SRBSDV-RRSV synergism promoted replication and movement of both viruses and inhibited the host immunity by enhancing the gene suppressing effect exerted by one of them (RRSV).

Inhibition of miR-274-3p increases BmCPV replication by regulating the expression of BmCPV NS5 gene in Bombyx mori.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the major pathogens that pose a big challenge to the sericulture industry. Growing evidences have shown that microRNAs play key roles in the regulations of host-pathogen interactions in insects. MicroRNAs have been found in silkworms, whether and how they affect the silkworm-BmCPV interactions are still unknown. Here we investigate the effect of miR-274-3p on the BmCPV replication in the BmCPV-infected silkworm larvae. In our study, BmCPV Nonstructural protein 5 (NS5) was identified to be the target of miR-274-3p based on bioinformatics analysis and luciferase reporter assay. The abundance of NS5 was significantly increased in the presence of miR-274-3p inhibitor based on the qRT-PCR and Western blotting results. Further, qRT-PCR results revealed that the expression of polyhedrin gene of BmCPV in the larvae after applying miR-274-3p inhibitor was significantly increased comparing with that of larvae with negative control. Our results suggest that inhibition of miR-274-3p could facilitate BmCPV replication by up-regulating BmCPV NS5 gene expression and are insightful for further exploring the interactions between silkworm and BmCPV.

Tubules of plant reoviruses exploit tropomodulin to regulate actin-based tubule motility in insect vector.

Plant reoviruses are known to exploit virion-packaging tubules formed by virus-encoding non-structural proteins for viral spread in insect vectors. Tubules are propelled by actin-based tubule motility (ABTM) to overcome membrane or tissue barriers in insect vectors. To further understand which insect factors mediate ABTM, we utilized yeast two-hybrid and bimolecular fluorescence complementation assays to test interactions between tubule protein Pns10 of rice dwarf virus (RDV), a plant reovirus, and proteins of its insect vector, the leafhopper Nephotettix cincticeps. Tropomodulin (Tmod), vitellogenin, and lipophorin precursor of N. cincticep displayed positive and strong interaction with Pns10, and actin-associated protein Tmod interacted with Pns10 in pull-down assay and the co-immunoprecipitation system. Further, we determined Pns10 tubules associated with Tmod in cultured cells and midgut of N. cincticep. The expression dynamic of Tmod was consistent with that of Pns10 and the fluctuation of RDV accumulation. Knockdown of Tmod inhibited the Pns10 expression and viral accumulation, thus decreasing the viruliferous rates of leafhopper. These results suggested that Tmod was involved in viral spread by directly interacting with Pns10 tubules, finally promoting RDV infection. This study provided direct evidence of plant reoviruses utilizing an actin-associated protein to manipulate ABTM in insect vectors, thus facilitating viral spread.

A mitochondrial membrane protein is a target for rice ragged stunt virus in its insect vector.

Rice ragged stunt virus (RRSV; Reoviridae) is exclusively transmitted by the brown planthopper Nilaparvata lugens in a persistent-propagative manner. It is understood that RNA viral proliferation is associated with the intracellular membranes of the insect host cells. However, the molecular mechanisms of the interaction between the RRSV proliferation and the intracellular membranes remain essentially unknown. It will be of great interest to determine whether RRSV protein(s) directly interact with intracellular membrane components of its host cells. In this study, we identified a RRSV nonstructural protein Pns10 interacting with a host oligomycin-sensitivity conferral protein (OSCP) using yeast two-hybrid system. The interaction between RRSV Pns10 and N. lugens OSCP was verified by a glutathione S-transferase pull-down assay. Confocal miscopy revealed colocalization of these two proteins in the cytoplasm of the salivary gland cells during the viral infection. The virions were further detected in the mitochondria under confocal miscopy and transmission electron microscopy combined with western blotting assay. This is the first observation that RRSV protein has a direct link with mitochondria. Suppressing OSCP gene expression by RNA interference notably decreased the viral loads in RRSV-infected insects. These findings revealed novel aspects of a viral protein in targeting the host mitochondrial membrane and provide insights concerning the mitochondrial membrane protein-based virus proliferation mode in the insect vector.

The speed of tubule formation of two fijiviruses corresponds with their dissemination efficiency in their insect vectors.

BACKGROUND: Rice black-streaked dwarf virus (RBSDV) and Southern rice black-streaked dwarf virus (SRBSDV) are two closely related fijiviruses transmitted by the small brown planthopper (SBPH) and white-backed planthopper (WBPH), respectively. SRBSDV has a latent period 4 days shorter than that of RBSDV, implying a more efficient spread in insect vector. Currently, the mechanisms underlying this higher efficiency are poorly understood. However, our recent studies have implicated a role of virus induced tubular structures in the dissemination of fijiiruses within their insect vectors. METHODS: Immunofluorescence labeling was performed to visualize and compare the dynamics of P7-1 tubule formation of the RBSDV and SRBSDV in their own vector insects and nonhost Spodoptera frugiperda (Sf9) cells. RESULTS: Tubule formation of SRBSDV P7-1 was faster than that of RBSDV P7-1. For RBSDV, P7-1 formed tubules were observed at 3-days post-first access to diseased plants (padp) in SBPH. For SRBSDV, these structures were detected as early as 1 day padp in WBPH. Importantly, similar phenomena were observed when P7-1 proteins from the two viruses were expressed alone in Sf9 cells. CONCLUSIONS: Our research revealed a relationship between the speed of P7-1 tubule formation and virus dissemination efficiency and also supports a role of such tubular structures in the spread of reoviruses within insect vectors.

Transcriptome analysis of interactions between silkworm and cytoplasmic polyhedrosis virus.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects silkworm midgut (MG) and multiplication occurs mainly in posterior midgut (PM). In this study, MG and fat body (FB) were extracted at 0, 3, 24, and 72 h after BmCPV infection. The total sequence reads of each sample were more than 1510000, and the mapping ratio exceeded 95.3%. Upregulated transcripts increased in MG during the infection process. Gene ontology (GO) categories showed that antioxidants were all upregulated in FB but not in MG. BGI001299, BGI014434, BGI012068, and BGI009201 were MG-specific genes with transmembrane transport function, the expression of which were induced by BmCPV. BGI001299, BGI014434, and BGI012068 expressed in entire MG and may be involved in BmCPV invasion. BGI009201 expressed only in PM and may be necessary for BmCPV proliferation. BmPGRP-S2 and BGI012452 (a putative serine protease) were induced by BmCPV and may be involved in immune defense against BmCPV. The expression level of BmCPV S1, S2, S3, S6, and S7 was high and there was no expression of S9 in MG 72 h, implying that the expression time of structural protein coding genes is earlier. These results provide insights into the mechanism of BmCPV infection and host defense.

Small interfering RNA pathway modulates persistent infection of a plant virus in its insect vector.

Plant reoviruses, rhabdoviruses, tospoviruses, and tenuiviruses are transmitted by insect vectors in a persistent-propagative manner. How such persistent infection of plant viruses in insect vectors is established and maintained remains poorly understood. In this study, we used rice gall dwarf virus (RGDV), a plant reovirus, and its main vector leafhopper Recilia dorsalis as a virus-insect system to determine how the small interference (siRNA) pathway modulates persistent infection of a plant virus in its insect vector. We showed that a conserved siRNA antiviral response was triggered by the persistent replication of RGDV in cultured leafhopper cells and in intact insects, by appearance of virus-specific siRNAs, primarily 21-nt long, and the increased expression of siRNA pathway core components Dicer-2 and Argonaute-2. Silencing of Dicer-2 using RNA interference strongly suppressed production of virus-specific siRNAs, promoted viral accumulation, and caused cytopathological changes in vitro and in vivo. When the viral accumulation level rose above a certain threshold of viral genome copy (1.32 × 10(14) copies/µg insect RNA), the infection of the leafhopper by RGDV was lethal rather than persistent. Taken together, our results revealed a new finding that the siRNA pathway in insect vector can modulate persistent infection of plant viruses.

Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus.

UNLABELLED: Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/µg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. IMPORTANCE: Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an incompetent vector, the small brown planthopper (SBPH). Here, we show that silencing of the core component Dicer-2 of the small interfering RNA (siRNA) pathway increases viral titers in the midgut epithelium past the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/µg of midgut RNA) for viral dissemination into the midgut muscles and then into the salivary glands, allowing the SBPH to become a competent vector of SRBSDV. This result is the first evidence that the siRNA antiviral pathway has a direct role in the control of viral dissemination from the midgut epithelium and that it affects the competence of the virus's vector.

Quantification of southern rice black streaked dwarf virus and rice black streaked dwarf virus in the organs of their vector and nonvector insect over time.

Southern rice black streaked dwarf virus (SRBSDV) and rice black streaked dwarf virus (RBSDV) are serious rice-infecting reoviruses, which are transmitted by different planthoppers in a persistent propagative manner. In this study, we quantitatively compared the spatial distribution of SRBSDV and RBSDV contents over time in their vector and nonvector insects using real time-PCR. Genome equivalent copies (GEC) were assessed every 2 days from 0 to 14 days after a 3-days acquisition access period (AAP) on infected plants. Results revealed 293.2±21.6 to 404.1±46.4 SRBSDV GEC/ng total RNA in whole body of white-backed planthopper (WBPH, Sogatella furcifera) at day 0 and 12 and 513.5±88.4 to 816.8±110.7 RBSDV GEC/ng total RNA in the whole body of small brown planthopper (SBPH, Laodelphax striatellus) at day 0 and 14, respectively, after 3-days AAP. Highest GEC of both viruses were found in the gut of their respective vectors. Although SRBSDV was detected in the gut of SBPH, it did not spread into the hemolymph or other organs. After an 8-day latent period, the transmission efficiency of SRBSDV and RBSDV by their respective vectors was significantly positively correlated with GEC in the salivary gland (r(2)=0.7808, P=0.0036 and r(2)=0.9351, P<0.0001, respectively, at α=0.05). Together, these results confirm that accumulation of >200 SRBSDV or RBSDV GEC/ng total RNA in the gut of vector, indicated threshold for further spread and the virus content in the salivary gland was significantly correlated with transmission efficiency by their respective vectors.

A novel, in vivo, indoor method to preserve rice black-streaked dwarf virus in small brown planthopper using wheat seedling as a bridge host.

Rice black-streaked dwarf virus (RBSDV) naturally infects Gramineae plants through small brown planthopper (SBPH) as a vector. However, RBSDV cannot be transmitted to the SBPH offspring through transovarian transmission. Wheat plant, an important intermediate host in winter, is essential for the completion of the annual cycle of RBSDV in farm ecosystem. We developed a novel, in vivo, indoor method to preserve RBSDV in SBPH using wheat seedling as a bridge host. The temperature range of 23-27°C was initially selected to rear the insects and plants. Before initiating the scheme cycle, viruliferous SBPH was obtained by feeding the virus-free 1st to 2nd instar nymphs with RBSDV-infected rice plants. Four to six RBSDV-infected SBPH were placed per plant to inoculate wheat seedlings at two-to-four leaf stages. After 48 h of inoculation, the viruliferous SBPH were removed. Five mated, newly emerged virus-free SBPH females were then transferred onto each inoculated plant and allowed to lay eggs for 48 h. The newly hatched SBPH were raised on wheat seedlings until the 2nd instar nymph stage, and then transferred onto healthy rice seedlings for further development until 5th instar nymphs or adults. These newly obtained viruliferous SBPH can be used for inoculating new wheat seedlings in the succeeding maintenance cycles, or for further experiments. We discovered that the incubation period of RBSDV in wheat seedlings synchronized with the gestation period of SBPH eggs at four to six inoculated viruliferous SBPH per plant and lasted for approximately seven days. In addition, this period was optimal for enhancing the SBPH infection ratio because SBPH nymphs can only acquire the virus after they hatch. The RBSDV infection ratio of the SBPHs acquired through this method consistently exceeded 50%.

Significant inhibition of two different genotypes of grass carp reovirus in vitro using multiple shRNAs expression vectors.

The hemorrhagic disease of grass carp (Ctenopharyngodon idellus), caused by grass carp reovirus (GCRV), is the most severe disease of the fish that leads to huge economic losses. GCRV, belonging to the genus Aquareovirus of the family Reoviridae, has been classified into three genotypes based on their phylogenetic relationship. It is essential to develop an effective method to inhibit the replication of different genotypes of GCRV simultaneously. In this report, two multiple-shRNAs expression vectors, named pMultishVP2/2 and pMultishVP6/7, were generated and investigated. pMultishVP2/2 targeted the VP2 gene of GCRV-JX0901 (genotype I) and the VP2 gene of HGDRV (Hubei grass carp disease reovirus; genotype III). pMultishVP6/7 targeted the VP7 gene of GCRV-JX0901 and the VP6 gene of HGDRV. These two multiple-shRNAs expression vectors can simultaneously, significantly inhibit the replication of GCRV-JX0901 and HGDRV in vitro. Compared to the positive control, CPE induced by GCRV-JX0901 or HGDRV in cell transfected with shRNA transcribing vector was significantly delayed. The quantitative PCR analysis of the GCRV genomic RNA revealed that the pMultishVP2/2 could simultaneously inhibit the GCRV-JX0901 and HGDRV VP2 coding genes by 89.02% and 89.84%, respectively. The pMultishVP6/7 could simultaneously inhibit the GCRV-JX0901 VP7 coding gene and HGDRV VP6 coding gene by 80.63% and 86.78%, respectively. Furthermore, compared to the positive control, the indirect immunofluorescence assay and western blot demonstrated that the protein expression of the two genotypes of GCRV decreased significantly. The results in this study indicated that this multiple-shRNAs expression system could be used as a cross-reactive antiviral agent for treating the hemorrhagic disease of grass carp caused by multiple genotypes of GCRV.

Development of an Ussuri catfish Pseudobagrus ussuriensis skin cell line displaying differential cytopathic effects to three aquatic animal viruses.

An Ussuri catfish Pseudobagrus ussuriensis skin (UCS) cell line was developed and subcultured for more than 60 passages. UCS cells consisted of mostly epithelial-like cells and multiplied well in TC199 medium supplemented with 10% fetal bovine serum at 25°C. Chromosome analysis revealed that most UCS cells had a normal diploid karyotype with 2n=52. UCS cells showed differential cytopathic effects (CPEs) after inoculation of spring viremia of carp virus (SVCV, a negative-strand RNA virus), grass carp reovirus (GCRV, a multi-segmented double-stranded RNA virus) and Rana grylio virus (RGV, a large double-stranded DNA virus), and were indicative of high sensitivities to these three aquatic animal viruses by a virus titration study. The CPE caused by SVCV appeared as rounded and granular cells, grape-like clusters and small lytic plaques. Characteristic CPE containing plaque-like syncytia was induced by GCRV. RGV-infected cells produced typical CPE characterized by cells shrinkage and aggregation, formation of clear plaques and cell sheet detachment. Furthermore, significant fluorescent signals were observed after UCS cells were transfected with green fluorescent protein reporter plasmids, and the development of CPE induced by a recombinant RGV, ΔTK-RGV, in UCS cells was illustrated using a combination of light and fluorescence microscopy. The data from this study suggested that UCS cell line can potentially serve as a useful tool for the comparison study of different aquatic animal viruses and the isolation of some newly emerging viruses in Ussuri catfish farming.

Development of continuous cell culture of brown planthopper to trace the early infection process of oryzaviruses in insect vector cells.

UNLABELLED: Rice ragged stunt virus (RRSV), an oryzavirus in the family Reoviridae, is transmitted by the brown planthopper, Nilaparvata lugens, in a persistent-propagative manner. Here, we established a continuous cell line of brown planthopper to investigate the mechanism underlying the formation of the viroplasm, the putative site for viral replication and assembly, during infection of RRSV in its insect vector cells. Within 24 h of viral infection of cultured cells, the viroplasm had formed and contained the viral nonstructural proteins Pns6 and Pns10, known to be constituents of viroplasm. Core capsid protein P3, core particles, and newly synthesized viral RNAs were accumulated inside the viroplasm, while outer capsid protein P8 and virions were accumulated at the periphery of the viroplasm, confirming that the viroplasm induced by RRSV infection was the site for viral replication and assembly. Pns10 formed viroplasm-like inclusions in the absence of viral infection, suggesting that the viroplasm matrix was largely composed of Pns10. Pns6 was recruited in the viroplasm by direct interaction with Pns10. Core capsid protein P3 was recruited to the viroplasm through specific association with Pns6. Knockdown of Pns6 and Pns10 expression using RNA interference inhibited viroplasm formation, virion assembly, viral protein expression, and viral double-stranded RNA synthesis. Thus, the present study shows that both Pns6 and Pns10 of RRSV play important roles in the early stages of viral life cycle in its insect vector cells, by recruiting or retaining components necessary for viral replication and assembly. IMPORTANCE: The brown planthopper, a commonly distributed pest of rice in Asia, is the host of numerous insect endosymbionts, and the major vector of two rice viruses (RRSV and rice grassy stunt virus). For the first time, we successfully established the continuous cell line of brown planthopper. The unique uniformity of brown planthopper cells in the monolayer can support a consistent, synchronous infection by endosymbionts or viral pathogens, improving our understanding of molecular insect-microbe interactions.