Archives of Virology

, Volume 157, Issue 4, pp 661–668

Avian reovirus triggers autophagy in primary chicken fibroblast cells and Vero cells to promote virus production

Authors

    • Ministry of Education Key Lab for Avian Preventive MedicineCollege of Veterinary Medicine, Yangzhou University
    • College of Bioscience and BiotechnologyYangzhou University
  • Ke Jiang
    • Ministry of Education Key Lab for Avian Preventive MedicineCollege of Veterinary Medicine, Yangzhou University
  • Xiaorong Zhang
    • Ministry of Education Key Lab for Avian Preventive MedicineCollege of Veterinary Medicine, Yangzhou University
  • Miao Zhang
    • College of Bioscience and BiotechnologyYangzhou University
  • Zhizhi Zhou
    • College of Bioscience and BiotechnologyYangzhou University
  • Maozhi Hu
    • College of Bioscience and BiotechnologyYangzhou University
  • Rui Yang
    • Ministry of Education Key Lab for Avian Preventive MedicineCollege of Veterinary Medicine, Yangzhou University
  • Chenli Sun
    • Ministry of Education Key Lab for Avian Preventive MedicineCollege of Veterinary Medicine, Yangzhou University
    • Ministry of Education Key Lab for Avian Preventive MedicineCollege of Veterinary Medicine, Yangzhou University
Original Article

DOI: 10.1007/s00705-012-1226-x

Cite this article as:
Meng, S., Jiang, K., Zhang, X. et al. Arch Virol (2012) 157: 661. doi:10.1007/s00705-012-1226-x

Abstract

Avian reovirus (ARV) is an important cause of disease in poultry. Although ARV is known to induce apoptosis in infected cells, the interaction between ARV and its target cells requires further elucidation. In this report, we show that the ARV isolate strain GX/2010/1 induces autophagy in both Vero and primary chicken embryonic fibroblast (CEF) cells based on the appearance of an increased number of double-membrane vesicles, the presence of GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) dot formation, and the elevated production of LC3II. We further demonstrate that the class I phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway contributes to autophagic induction by ARV infection. Moreover, treatment of ARV-infected cells with the autophagy inducer rapamycin increased viral yields, while inhibition of the autophagosomal pathway using chloroquine led to a decrease in virus production. Altogether, our studies strongly suggest that autophagy may play a critical role in determining viral yield during ARV infection.

Introduction

Avian reoviruses (ARVs) are members of the genus Orthoreovirus, one of the 12 genera composing the family Reoviridae [1]. ARVs are non-enveloped viruses that contain 10 double-stranded RNA genome segments and replicate in the cytoplasm of infected cells. Unlike their mammalian counterparts, ARVs cause massive fusion of host cells but lack hemagglutination activity [2]. Infection by ARVs is associated with a variety of disease conditions, including viral arthritis, chronic respiratory diseases, and malabsorption syndrome. Although apoptosis (type I programmed cell death, or PCD) is a critical strategy utilized by several ARV strains, including S1133 [3], for its pathological effects, the role of autophagy (type II PCD) in ARV-related pathogenesis has not been reported.

Autophagy is an evolutionarily conserved process involving sequestration of the cytoplasm and organelles into double-membrane vesicles (autophagosomes) that traffic their contents to the lysosomes, where recycling occurs [4]. During the formation of the autophagosome membrane, microtubule-associated protein-1 light-chain 3 (LC3) is converted from a free (LC3 I) to a phosphatidylethanolamine-conjugated form (LC3 II). The accumulation of LC3 II and its localization to the autophagosome (punctate dot formation) are commonly used as markers for autophagy. The normal physiological function of autophagy is the removal of long-lived proteins and damaged organelles, but in addition to its role in cellular homeostasis, autophagy can also be a form of PCD. Autophagy is tightly regulated by cellular signaling pathways. A major negative regulator of autophagy is the serine/threonine kinase mammalian target of rapamycin (mTOR). The class I phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway inhibits autophagic induction, whereas the class III PI3K/Beclin-1 pathway positively regulates autophagy [5]. Although autophagy was originally recognized as a response to starvation, it is also triggered by stress stimuli, such as oxidative stress, chemicals, and infection by intracellular pathogens, including viruses. Increasing evidence indicates that the autophagosomal structures induced by many RNA viruses may provide a scaffold for virus replication [6, 7]. However, the extent to which autophagy is induced during ARV infection remains largely unknown.

Here, we report the discovery of an ARV isolate, strain GX/2010/1, that induces autophagy in both Vero cells and primary chicken embryonic fibroblast (CEF) cells. Additional results, obtained from the chemical stimulation or inhibition of the autophagy process, indicate that autophagy plays a favorable role in virus propagation.

Materials and methods

Cell lines, virus and plasmid

Vero (African green monkey kidney) cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C in a 5% CO2 atmosphere. CEF cells from 10-day-old specific-pathogen-free eggs were grown in M199 medium supplemented with 3% FBS. The complete genomic sequence for the avian reovirus strain GX/2010/1 was deposited in GenBank (accession numbers JN559375, JN559376, JN559377, and JN559378). ARV GX/2010/1 was propagated and titrated in CEF cells. Plasmid expressing GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) was purchased from Origene.

Antibodies and reagents

The antibody to cleaved caspase-3, the phospho-specific antibodies to mTOR (Ser2448) and Akt (Thr308) and total antibodies against mTOR and Akt were purchased from Cell Signaling Technology. Polyclonal rabbit anti-LC3 and monoclonal antibody against β-actin were purchased from Sigma. Monoclonal antibody to Sequestosome-1 (p62) was purchased from Epitomics. Rapamycin and chloroquine (CQ) were obtained from Sigma.

Virus infection and inactivation of virus by heat

Vero and CEF cells were infected with ARV at a multiplicity of infection (MOI) of 10 and 1, respectively, or sham infected with phosphate-buffered saline (PBS), at 37°C for 1 h in serum-free DMEM (Vero) or M199 (CEF). The cells were then washed three times with PBS and incubated at 37°C in DMEM or M199 supplemented with 1% FBS. To modulate the induction of autophagy, cells were treated with rapamycin or CQ for 1 h prior to virus infection. Subsequently, the cells were infected with ARV in the presence or absence of the compounds. For experiments designed to determine the virus yield, Vero or CEF cells were infected with ARV at an MOI of 0.01, and multi-step viral growth curves were determined as described previously [8]. Heat inactivation of ARV was achieved by heating for 20 min at 60°C as described by Pandha et al. [9].

Cell transfection and fluorescence microscopy

Vero or CEF cells were transfected with a plasmid expressing GFP-LC3 using the Lipofectamine 2000 according to the manufacturer’s instructions. Dot formation by GFP-LC3 was detected under a fluorescence microscope (BX50, Olympus) following drug treatment and/or ARV infection. Transfected cells were considered to have accumulated autophagosomes when five or more puncta were observed, because no more than four puncta were found in mock-infected cells. A total of 100 transfected cells were examined per well, in triplicate, from three independent experiments.

Transmission electron microscopy

For ultrastructural analysis, standard transmission electron microscopy (TEM) was carried out on virus-infected Vero cells. Cells were collected 24 hours postinfection and fixed in 2.5% glutaraldehyde for at least 3 h. Thin sections (90 nm) were cut and examined at 80 kV using a JEOL 1200EX transmission electron microscope. Approximately 15 cells were counted; autophagosomes were defined as double-membrane vacuoles measuring 1.0 to 2.0 μm.

Immunoblot analysis

Vero or CEF cells in 60-mm dishes were infected with ARV at an MOI of 10 and 1, respectively. One hour after infection, the medium was removed and replaced with DMEM or M199 supplemented with 1% FBS for the duration of the experiment. Virus-infected cells were harvested at the times indicated, and immunoblot (IB) was performed essentially as described previously [8]. The expression of LC3II and β-actin was determined using a calibrated GS-670 densitometer. All IB experiments were performed twice.

Real-time quantitative RT-PCR

Real-time quantitative RT-PCR (qRT-PCR) analysis of the σC gene of ARV and the housekeeping gene β-actin were performed as described previously [20]. Total RNA was extracted from infected cells using an RNeasy Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. For the qRT-PCR assay, amplification and fluorescence detection were performed on an Applied Biosystems 7500 real-time PCR cycler. The primers used for the ARV σC gene were 5′-att gtt gtt cat tgg gat gg-3′ and 5′-tcc cag cac ggc gcc aca cc-3′. The primers used for β-actin were 5′-agt act ccg tgt gga tcg gc-3′ and 5′-gct gat cca cat ctg ctg ga-3′. The TaqMan® reaction assay was optimized using a SuperScript™ III Platinum® One-Step qRT-PCR Kit (Invitrogen) using a final concentration of 400 nM for each of the σC gene primers and 200 nM Taqman probe in a total reaction volume of 25 μl containing 2 μl of extracted RNA. After an initial incubation of 10 min at 95°C, 40 cycles (95°C for 1 min and 55°C for 1 min) were performed for amplification. The specificity of amplification was confirmed by melting curve analysis. Each sample was run in triplicate. After ensuring that amplification efficiencies were the same, the expression of the σC gene was normalized relative to that of the housekeeping gene. The final results were expressed as normalized σC gene RNA levels.

Statistical analysis

The data were evaluated using Student’s t-test and the SPSS V17.0 software (SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant at p < 0.05.

Results

ARV triggers autophagosome formation in Vero cells

To examine whether the ARV isolate strain GX/2010/1 induces apoptosis, Vero cells were infected with ARV at an MOI of 10 for different amounts of time, and cleavage of caspase-3 was analyzed by immunoblotting. Caspase-3 is the major effector of apoptosis. As shown in Fig. 1A, the cleaved, active fragment of caspase-3 was first observed at 24 h postinfection (hpi). Strong accumulation of cleaved caspase-3 in ARV-infected Vero cells was further detectable from 48 to 120 hpi, indicating that ARV GX/2010/1 induces apoptosis in both the middle and late stages of infection.
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Fig. 1

Avian reovirus triggers autophagy in Vero cells. (A) Vero cells were infected with avian reovirus (ARV) strain GX/2010/1 at a multiplicity of infection (MOI) of 10, and at the indicated time points, cell lysates were analyzed by immunoblot (IB) assay with a specific antibody to the activated form of caspase-3. The β-actin protein level was used as a loading control. (B) Vero cells were transfected with GFP-LC3, followed by ARV infection. The number of cells with punctate GFP-LC3 is displayed as a histogram. The pictures show mock-infected cells, cells treated with rapamycin for 24 h as a positive control, and cells infected with ARV for 24 h (** p < 0.01). (C) Transmission electron microscopy analysis. a, Untreated control cells showing normal distribution of organelles. b, Cup-shaped membranous structures in the cytoplasm (indicated by the arrow). c, A typical autophagosome and a swollen mitochondrion; a virus particle (V) can also be observed in the same cell. d, A typical autophagosome containing organelles undergoing degenerative changes; the two limiting membranes of the autophagosome (indicated by the arrows) are visible at the upper rim of the vacuole. e, An initial autophagosome (AVi), containing electron-dense myelin figures and other organelles, shown docking and fusing with a lysosome (L) to form a single-membrane vacuole. f, The degradation of the engulfed rough ER is advanced in this degradative autophagosome (AVd), where the remnants of the ribosomes form electron-dense and partially amorphous masses (indicated by R). This AV has additionally fused with a multivesicular endosome, as indicated by the contents of the numerous small vesicles observed (indicated by arrows). (D) The time course induction of LC3II in ARV-infected Vero cells was investigated by immunoblot (IB). R stands for rapamycin. Cells treated with rapamycin were used as the positive control. Sample loading was controlled by use of an anti-β-actin antibody as indicated. Densitometry was performed for quantification, and the ratios of LC3II to β-actin are presented below the blots. The results shown are representative of two separate experiments. (E) Vero cells were treated as in D, and p-62 expression was determined by IB. (F) ARV was inactivated by heating for 20 min at 60 °C and then used to infect Vero cells at the indicated times. Induction of LC3II was determined as in D. All IB experiments were performed twice

It is known that both autophagy and apoptosis are triggered following infection by several kinds of viruses. To determine whether autophagy is induced upon ARV GX/2010/1 infection, Vero cells were transfected with a plasmid expressing the GFP-LC3 fusion protein, an autophagy marker, followed by ARV infection for 24 h. Rapamycin, an autophagy inducer, was used as a positive control. As shown in Fig. 1B, punctate GFP-LC3 proteins accumulated in both rapamycin-treated and ARV-infected cells. Cells with five or more LC3 dots in the cytoplasm were counted as cells with autophagosome formation. We observed that more dots appeared in both virus-infected and rapamycin-treated cells than in control cells (p < 0.01) (Fig. 1B).

To further confirm that autophagy is induced by ARV infection, transmission electron microscopy was performed on mock-treated or ARV-infected cells. As depicted in Fig. 1C, infected cells exhibited several double-membrane vesicles (autophagosomes) that were not present in control cells. In addition, we also observed cup-shaped membranous structures (Fig. 1C, b), virus particles in direct association with some ultrastructures (Fig. 1C, c), single-membrane vacuoles (Fig. 1C, e), and other ultrastructures in ARV-infected cells.

LC3 lipidation is considered to be a hallmark of autophagy induction, making the ratio of LC3II to actin an accurate indicator for determining the presence of autophagy. Fig. 1D shows that, relative to uninfected (mock) cells, conversion of endogenous LC3 to LC3II was markedly induced in Vero cells at 24 h after infection, with peak induction occurring at 48 h. These increased levels of LC3II production were sustained for up to 96 hpi. Increased LC3II production was also detected in rapamycin-treated cells.

The observed increase in LC3 conversion and accumulation of punctate LC3 dots in ARV-infected Vero cells are both early events in the autophagy pathway. To examine whether a complete autophagic response (i.e., an autophagic flux) was induced by ARV, we determined the levels of the autophagic substrate, p62, which is specifically degraded by the autophagic-lysosomal pathway. Fig. 1E illustrates that increased expression of p62 was detected at 12 and 24 hpi but that p62 expression in infected cells was undetectable at 48 h and all subsequent time points. These results indicate that ARV can induce a complete autophagic response in Vero cells.

To investigate whether viral replication is involved in ARV-induced autophagy, Vero cells were treated with heat-inactivated ARV for various times. As shown in Fig. 1F, the conversion of endogenous LC3 to LC3II was largely undetectable in infected cells at all of the time points examined. Rapamycin-treated cells, however, displayed markedly increased LC3II production across all time points measured.

Autophagy is induced in ARV-infected CEF cells

To explore whether ARV can trigger autophagy in ARV-infected primary CEF cells, both conversion of LC3 and LC3 dot formation were investigated. As shown in Fig. 2A, in cells transfected with GFP-tagged LC3, LC3-GFP redistribution into discrete dots was significantly increased by ARV infection and rapamycin treatment. Fig. 2B shows that the expression level of LC3II was markedly increased at 12 hpi, reaching its highest level at 16 hpi. Moreover, the expression of LC3II was not observed in ARV-infected CEF cells (Fig. 2C). Taken as a whole, these results indicate that ARV induces autophagy in primary CEF cells.
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Fig. 2

Autophagy is induced in primary chicken embryonic fibroblast (CEF) cells. (A) CEF cells were transfected with GFP-LC3, followed by ARV infection (MOI of 1). Formation of GFP-LC3 puncta was investigated as described in Fig. 1B (** p < 0.01). (B) CEF cells were infected with avian reovirus (MOI of 1) as described in Fig. 1D, and the time course of induction of LC3II was investigated by immunoblot (IB). R stands for rapamycin. (C) CEF cells were treated with heat-inactivated ARV at the times indicated, and LC3II production was measured by IB. All IB experiments were performed twice

The class I PI3K/Akt/mTOR pathway contributes to ARV-triggered autophagy

To determine which signaling pathway is responsible for the induction of autophagy in ARV-infected Vero cells, activation of the class I PI3K/Akt/mTOR, class III PI3K/Beclin-1, and MAPK pathways were examined by immunoblotting. The inhibitor of mTOR rapamycin was used as a control. As shown in Fig. 3A, ARV infection resulted in a significant reduction of mTOR phosphorylation at the time points examined (p < 0.01). Furthermore, phosphorylation of Akt was also reduced in ARV-infected cells (Fig. 3B). However, the expression of Beclin-1 was not observed after ARV infection (data not shown). These results indicate that the class I PI3K/Akt/mTOR pathway contributes to ARV-triggered autophagy in Vero cells.
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Fig. 3

The class I PI3K/Akt/mTOR pathway contributes to ARV-triggered autophagy. Vero cells were infected with ARV (MOI of 10) at the indicated times. Cell lysates were used to determine the activation of mammalian target of rapamycin (mTOR) and Akt with specific antibodies. Upper panel, immunoblot (IB) analysis; lower panel, quantification of the phosphorylated bands (** p < 0.01). (A) Reduced levels of phosphorylation of mTOR. (B) Decreased activation of Akt. All IB experiments were performed in triplicate (** p < 0.01)

Regulation of autophagy induction affects virus yield in ARV-infected Vero and CEF cells

To investigate whether autophagic induction during ARV infection is a host antiviral response or a viral replication mechanism, we tested the effects of the autophagy modulators rapamycin and chloroquine (CQ) on both viral replication and virus yield. Rapamycin has been shown to induce autophagy by inhibiting the mTOR pathway. CQ inhibits the autophagic process by preventing lysosome-autophagosome fusion and the subsequent degradation of the contents of the autophagosome [10], which leads to a marked accumulation of autophagic vacuoles [11]. As LC3II is associated with autophagic vacuoles, CQ treatment also induces intense LC3II accumulation [12]. The effective concentration of these compounds was determined by dose-response assays to prevent cytotoxicity (data not shown). It was found that neither rapamycin nor CQ had any effect on cell viability at the concentrations used in this study (data not shown). Each chemical was added to Vero cells, in separate experiments, 30 min prior to ARV infection. We first measured the production of LC3II upon treatment with these compounds in ARV-infected Vero and CEF cells. As illustrated in Fig. 4A, for all of the time points examined, both rapamycin and CQ induced markedly increased production of LC3II in Vero cells infected with ARV (MOI of 10) compared to control infection. Of note, CQ treatment resulted in the production of the greatest amount of LC3II among the three treatments. Similar results were obtained in CEF cells (Fig. 4B). Next, the number of ARV σC gene copies expressed in Vero cells after infection was determined by qRT-PCR. Fig. 4C shows that σC gene copies in ARV-infected cells were significantly increased 24, 48 and 72 hpi after treatment with rapamycin. However, a significant reduction in σC gene copies was observed in cells treated with CQ. These data indicate that regulation of autophagy affects ARV replication (* p < 0.05; ** p < 0.01).
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Fig. 4

Regulation of ARV-induced autophagy affects viral yield. (A) and (B) Vero cells (A) and chicken embryonic fibroblast (CEF) cells (B) were pretreated with rapamycin (100 nM) or chloroquine (CQ) (50 μM) and then infected with ARV (MOI of 10 or 1) at the indicated times. LC3II production was then determined by immunoblot (IB). The ratios of LC3II to β-actin are presented below the blots. (C) Vero cells were treated as in A. Real-time PCR analysis of viral σC gene expression was performed (* p < 0.05; ** p < 0.01). (D) and (E) Vero cells (D) and CEF cells (* p < 0.05; ** p < 0.01). (E) were mock-treated or pretreated with rapamycin (100 nM) or CQ (50 μM) for 1 h. Cells were then infected with ARV at an MOI of 0.01, and viral yield was determined at the indicated times. All experiments were performed at least three times (* p < 0.05; ** p < 0.01)

Finally, we assessed the yield of virus in rapamycin- and CQ-treated Vero and CEF cells following ARV infection. As shown in Fig. 4D, ARV (MOI of 0.01) replicates well in infected Vero cells, with virus titers increasing as the infection time increased from 24 to 72 hpi. The virus yield increased approximately 1 log when cells were pre-treated with rapamycin. Conversely, when infected cells were pre-treated with CQ, virus yield decreased by nearly 1 log (** p < 0.01; * p < 0.05) (Fig. 4D). We observed similar effects of rapamycin and CQ on virus yield in CEF cells (** p < 0.01; * p < 0.05) (Fig. 4E). Together, these data indicate that enhanced autophagy correlates with increased viral yield in ARV-infected cells, whereas the inhibition of autophagy correlates with a decrease in the yield of ARV.

Discussion

A growing list of viruses has been shown to be involved in autophagy [13, 14]. It has been reported previously that rotavirus, a member of the family Reoviridae, can induce autophagic vacuoles via the NSP4 protein [15], suggesting that reoviruses might also trigger autophagy in infected cells. We show here that our ARV GX/2010/1 strain triggers autophagy in both Vero and primary CEF cells, as demonstrated by an increased number of double-membrane vesicles, increased GFP-LC3 dot formation, and the elevated production of LC3II. Notably, heat-inactivated ARV does not trigger autophagy in either Vero or CEF cells, suggesting that viral replication is necessary during autophagosome formation. In addition, our data indicate that the class I PI3K/Akt/mTOR pathway contributes to ARV-triggered autophagy. Moreover, use of the autophagy inducer rapamycin and the inhibitor CQ reveals that autophagy plays a positive role in ARV propagation. Taken together, our findings suggest that autophagy, as triggered by ARV, might be utilized for viral propagation.

It is well known that ARV induces apoptosis in primary CEF and several other cell lines, including Vero cells [1618]. Similar to the ARV S1133 strain, our ARV GX/2010/1 strain also induces apoptosis in Vero cells during the middle to late stages of infection. When comparing the time-course patterns for the induction of autophagy and apoptosis, it was found that ARV GX/2010/1 induced parallel autophagy and apoptosis at nearly the same time (from 24 to 120 hpi), indicating that both autophagy and apoptosis are induced upon ARV infection. However, it should be noted that the autophagic induction by ARV infection reached its peak at 48 and 72 hpi and declined at 96 hpi, while apoptotic induction was maintained at high levels from 72 to 120 hpi, suggesting an overlap between autophagic and apoptotic cell death during ARV infection. Together, our results provide new insights into the interaction between ARV and its target cells and suggest that autophagy might play a role in the pathogenesis of ARV infection as well as in apoptosis. Further research will be needed to elucidate the exact relationship between autophagy and apoptosis induced by ARV.

The induction of autophagy by different viruses is regulated by different signaling pathways. Besides its positive role in cell survival, the class I PI3K/Akt pathway has been shown to negatively regulate autophagy [19]. We observed that at the middle to late stages of ARV infection, activation of Akt and mTOR was inhibited in infected Vero cells, indicating a critical role of the PI3K/Akt/mTOR pathway in ARV-triggered autophagy. Interestingly, Lin et al. recently reported that the ARV strain S1133 activates the PI3K/Akt pathway during the early stages of infection (from 30 min to 2 h), resulting in delayed apoptosis in Vero cells [20]. However, in the same paper, Lin et al. further demonstrate that activation of Akt in ARV S1133-infected Vero cells was inhibited 4 h after infection [20]. Therefore, the data from our study concerning the time course of Akt activation is not contradictory to the related results from Lin et al., although they did not demonstrate activation of Akt during the middle to late stages of infection. Taken together, our work and that of others indicate that the class I PI3K/Akt pathway might play a critical role in both autophagy and apoptosis induced by ARV. Further research will need to be conducted to investigate whether other signaling pathways, such as the p53 pathway, are also involved in ARV-induced apoptosis [21]. Whether these pathways also participate in ARV-triggered autophagy will also need to be investigated in future work.

Although virus infection may induce autophagy, whether activation of the autophagic machinery can in turn enhance viral replication (as appears to be the case for poliovirus, mouse hepatitis virus, and others) or not (as in the case of vaccinia virus, herpes simplex virus type 1, etc.) appears to depend on the type of virus involved [19, 22]. Our findings not only demonstrate that ARV triggers autophagy but also that it enhances viral propagation in infected Vero and CEF cells. These observations are supported by the effects of autophagy-modulating compounds on both viral replication and virus yield. However, how autophagy affects ARV propagation remains unknown. To date, there is little in the literature to indicate whether other reoviruses, including mammalian reoviruses, can induce autophagy. Our data therefore provide a unique example of a reovirus that induces autophagy to enhance its own propagation.

Collectively, our results show that ARV induces autophagy both in Vero cells and in primary CEF cells and further suggest that autophagy may be beneficial for the propagation of ARV in infected cells. Our findings provide new insights into the mechanisms underlying the interaction between ARV and target cells.

Acknowledgments

This work was supported by grants from the State Key Laboratory of Veterinary Biotechnology of China (SKLVBF201109), the China Agriculture Research System (CARS-41-K08), and the Program for Changjiang Scholars and Innovative Research Teams in University (IRT0978).

Conflict of interest

None declared.

Copyright information

© Springer-Verlag 2012