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.

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.

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.

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).

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.

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.

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.

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.

Apoptosis induced by mammalian reovirus is beta interferon (IFN) independent and enhanced by IFN regulatory factor 3- and NF-κB-dependent expression of Noxa.

A variety of signal transduction pathways are activated in response to viral infection, which dampen viral replication and transmission. These mechanisms involve both the induction of type I interferons (IFNs), which evoke an antiviral state, and the triggering of apoptosis. Mammalian orthoreoviruses are double-stranded RNA viruses that elicit apoptosis in vitro and in vivo. The transcription factors interferon regulatory factor 3 (IRF-3) and nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) are required for the expression of IFN-ß and the efficient induction of apoptosis in reovirus-infected cells. However, it is not known whether IFN-ß induction is required for apoptosis, nor have the genes induced by IRF-3 and NF-κB that are responsible for apoptosis been identified. To determine whether IFN-ß is required for reovirus-induced apoptosis, we used type I IFN receptor-deficient cells, IFN-specific antibodies, and recombinant IFN-ß. We found that IFN synthesis and signaling are dispensable for the apoptosis of reovirus-infected cells. These results indicate that the apoptotic response following reovirus infection is mediated directly by genes responsive to IRF-3 and NF-κB. Noxa is a proapoptotic BH3-domain-only protein of the Bcl-2 family that requires IRF-3 and NF-κB for efficient expression. We found that Noxa is strongly induced at late times (36 to 48 h) following reovirus infection in a manner dependent on IRF-3 and NF-κB. The level of apoptosis induced by reovirus is significantly diminished in cells lacking Noxa, indicating a key prodeath function for this molecule during reovirus infection. These results suggest that prolonged innate immune response signaling induces apoptosis by eliciting Noxa expression in reovirus-infected cells.

Dynamics of Southern rice black-streaked dwarf virus in rice and implication for virus acquisition.

A novel viral disease of rice caused by Southern rice black-streaked dwarf virus (SRBSDV) has spread throughout East and Southeast Asia since the mid-2000s. Outbreaks of this viral disease occur yearly in southern parts of Japan concurrently with overseas migration of the planthopper vector Sogatella furcifera from southern China during the rainy season (from late June to early July). We examined the dynamics (changes in titer and localization) of SRBSDV on rice using reverse-transcription real-time polymerase chain reaction and determined the relationship between virus titer in plants and virus acquisition by S. furcifera. Under a constant temperature of 27°C, a substantial increase of SRBSDV titer in the leaf sheath together with typical symptoms (stunted growth and twisting of leaf tips) was observed at 20 days after the end of a 7-day exposure of viruliferous S. furcifera. Approximately 40% of S. furcifera acquired SRBSDV through feeding for 5 days on rice plants that were infected following exposure to viruliferous vectors for 10 to 15 days. These results suggest that rice infected by S. furcifera can be a source of SRBSDV before the next generation of S. furcifera emerges.

Host response to polyomavirus infection is modulated by RNA adenosine deaminase ADAR1 but not by ADAR2.

Adenosine deaminases acting on RNA (ADARs) catalyze the C-6 deamination of adenosine (A) to produce inosine (I), which behaves as guanine (G), thereby altering base pairing in RNAs with double-stranded character. Two genes, adar1 and adar2, are known to encode enzymatically active ADARs in mammalian cells. Furthermore, two size forms of ADAR1 are expressed by alternative promoter usage, a short (p110) nuclear form that is constitutively made and a long (p150) form that is interferon inducible and present in both the cytoplasm and nucleus. ADAR2 is also a constitutively expressed nuclear protein. Extensive A-to-G substitution has been described in mouse polyomavirus (PyV) RNA isolated late times after infection, suggesting modification by ADAR. To test the role of ADAR in PyV infection, we used genetically null mouse embryo fibroblast cells deficient in either ADAR1 or ADAR2. The single-cycle yields and growth kinetics of PyV were comparable between adar1(-/-) and adar2(-/-) genetic null fibroblast cells. While large T antigen was expressed to higher levels in adar1(-/-) cells than adar2(-/-) cells, less difference was seen in VP1 protein expression levels between the two knockout MEFs. However, virus-induced cell killing was greatly enhanced in PyV-infected adar1(-/-) cells compared to that of adar2(-/-) cells. Complementation with p110 protected cells from PyV-induced cytotoxicity. UV-irradiated PyV did not display any enhanced cytopathic effect in adar1(-/-) cells. Reovirus and vesicular stomatitis virus single-cycle yields were comparable between adar1(-/-) and adar2(-/-) cells, and neither reovirus nor VSV showed enhanced cytotoxicity in adar1(-/-)-infected cells. These results suggest that ADAR1 plays a virus-selective role in the host response to infection.

Reovirus inhibits poliovirus replication upon superinfection.

OBJECTIVE: Viral interference has been demonstrated in different systems, such as the effect of enterovirus infection on live-attenuated oral polio vaccine. In this study, the effect of reovirus which could exist in the human intestinal tract on poliovirus vaccine strains was investigated and could be an important factor to consider in oral polio vaccination. METHODS: Cells were infected with reovirus, then superinfected with poliovirus. The amount of viral yields was measured by the TCID(50) and plaque assay methods. Polioviral RNA synthesis was studied by real-time RT-PCR and the viral RNA load was calculated. Viral protein synthesis was determined using the techniques of immunoflourescent staining and PAGE followed by the immunoblotting experiment. RESULTS: Poliovirus superinfection of reovirus-infected cells resulted in inhibition of poliovirus replication. It was found that the inhibitory effect of reovirus was after establishment of its infection (2 h postinfection). There was no competition between the two viruses for cell attachment but poliovirus RNA and protein synthesis were inhibited. CONCLUSION: Infection of cells with reovirus could interfere with the growth of poliovirus upon superinfection. This phenomenon could be important to consider when using attenuated poliovirus vaccine.

Determinants of strain-specific differences in efficiency of reovirus entry.

Cell entry of reovirus requires a series of ordered steps, which include conformational changes in outer capsid protein µ1 and its autocleavage. The µ1N fragment released as a consequence of these events interacts with host cell membranes and mediates their disruption, leading to delivery of the viral core into the cytoplasm. The prototype reovirus strains T1L and T3D exhibit differences in the efficiency of autocleavage, in the propensity to undergo conformational changes required for membrane penetration, and in the capacity for penetrating host cell membranes. To better understand how polymorphic differences in µ1 influence reovirus entry events, we generated recombinant viruses that express chimeric T1L-T3D µ1 proteins and characterized them for the capacity to efficiently complete each step required for membrane penetration. Our studies revealed two important functions for the central δ region of µ1. First, we found that µ1 autocleavage is regulated by the N-terminal portion of δ, which forms an α-helical pedestal structure. Second, we observed that the C-terminal portion of δ, which forms a jelly-roll ß barrel structure, regulates membrane penetration by influencing the efficiency of ISVP* formation. Thus, our studies highlight the molecular basis for differences in the membrane penetration efficiency displayed by prototype reovirus strains and suggest that distinct portions of the reovirus δ domain influence different steps during entry.

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.

Ammonium chloride prevents lytic growth of reovirus and helps to establish persistent infection in mouse L cells.

Ammonium chloride, a lysosomotropic agent that raises intralysosomal pH, reduces the yield of reovirus during infection of mouse L cells. Subsequent removal of ammonium chloride results in the rapid establishment of a persistent infection.

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.

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.

The repression of cell wall- and plastid-related genes and the induction of defense-related genes in rice plants infected with Rice dwarf virus.

An analysis, using microarrays, of gene expression in rice plants infected with Rice dwarf virus revealed significant decreases in levels of expression of genes that are involved in the formation of cell walls, reflecting the stunted growth of diseased plants. The expression of plastid-related genes also was suppressed, as anticipated from the white chlorotic appearance of infected leaves. By contrast, the expression of defense- and stress-related genes was enhanced after viral infection. These results suggest that virus-infected rice plants attempt to survive viral infection and replication by raising the levels of expression of defense- and stress-related genes while suppressing the expression of genes required for the elongation of cells and photosynthesis.

Identification of the viral genes responsible for growth of strains of reovirus in cultured mouse heart cells.

Viral growth in specific tissue is usually required in order to lead to pathology. Two reovirus isolates (type 1 Lang and type 3 Dearing) differ in their capacity to grow in cultured mouse heart cells. The mammalian reoviruses contain a genome of 10 double-stranded RNA gene segments. By the use of 37 reassortant viruses (consisting of viruses with different combinations of genes derived from the two parents), difference in capacity of different strains to grow in heart cells was mapped to three different genes, all of which encode viral core proteins: the M1 gene (P less than 0.000044); the L1 gene (P = 0.00094); and the L3 gene (P = 0.019). Using the same set of reassortant viruses, the L1 (P = 0.00015) and L3 (P = 0.0065) genes were involved in differences of the ability of viral strains to grow in mouse L cells (fibroblasts), but the M1 gene (P = 0.12) was not. These findings suggest that the M1 gene plays an important and specific role in determining the relative capacity of certain viral strains to grow in the heart. Thus, we have identified viral genes responsible for differing growth capacity in heart muscle cells in culture. These findings provide a novel system for studies of viral myocarditis at a molecular genetic level.